Proteomic Analysis of Neonatal Mouse Brain: Evidence for Hypoxiaand Ischemia-Induced Dephosphorylation of Collapsin Response Mediator Proteins Yuan Zhou,† Inderjeet Bhatia,‡ Zhen Cai,† Qing-Yu He,§ Pik-To Cheung,‡ and Jen-Fu Chiu*,† Departments of Anatomy, Pediatrics and Adolescent Medicine, and Chemistry, The University of Hong Kong, Hong Kong Received February 9, 2008
Perinatal hypoxia and ischemia (HI) are a significant cause of mortality and morbidity. To understand the molecular mechanisms for HI-induced brain damage, here we used a proteomic approach to analyze the alteration and modification of proteins in neonatal mouse brain 24 h after HI treatment. Significant changes of collapsin response mediator proteins (CRMPs) were observed in HI brain. CRMPs are a family of cytosolic proteins involved in axonal guidance and neuronal outgrowth. We found that CRMP2, CRMP4 and CRMP5 proteins were altered post-translationally after HI treatment. Mass spectrometric and Western blot analyses detected hypophosphorylated CRMP proteins after HI. Further analysis of CRMP kinases indicated inactivation of cyclin dependent kinase 5 (CDK5), a priming kinase of CRMPs and a neuronal specific kinase that plays pivotal roles in neuronal development and survival. The reduction of CDK5 activity was associated with underexpression of its activator p35. Taken together, our findings reveal HI-induced dephosphorylation of CRMPs in neonatal brain and suggest a novel mechanism for this modification. Hypophosphorylated CRMPs might be implicated in the pathogenesis of HI-related neurological disorders. Keywords: collapsin response mediator proteins • hypoxia and ischemia • hypoxia-ischemia-induced brain damage • cyclin-dependent kinase 5 • p35
Introduction Hypoxia-ischemia (HI)-induced brain damage during the perinatal period is a leading cause of acute death and chronic neurological deficits in infants and children.1 Despite improvements in patient care over the past several decades, the incidence of HI encephalopathy, which is 0.2-0.8% in full-term infants or 60% in very premature neonates, remains essentially unchanged. While 20-50% of infants having HI encephalopathy die during the perinatal period, it is estimated that up to 25% of the survivors have neurological handicaps ranging from severe cerebral palsy to milder conditions such as mental retardation, learning disability and epilepsy.2 One major barrier to the prevention of these long-term deficits is the lack of understanding of the mechanism of encephalopathy. The molecular pathogenesis of HI-induced brain damage is heterogeneous. Interruption in placental blood flow is thought to be an underlying cause of brain damage. Reactive oxygen species generated as a result of perturbed cell metabolism are also known to mediate multiple pathological effects. In addition, neuronal necrosis and apoptosis play an important role * To whom correspondence should be addressed. Tel: 852-22990777. Fax: 852-28171006. E-mail:
[email protected]. † Department of Anatomy, The University of Hong Kong. ‡ Department of Pediatrics and Adolescent Medicine, The University of Hong Kong. § Department of Chemistry, The University of Hong Kong. 10.1021/pr800108k CCC: $40.75
2008 American Chemical Society
in the etiology of encephalopathy. However, it remains to be elucidated exactly how necrosis and apoptosis are induced after HI.1 One approach to derive new insight into the molecular mechanisms of HI-induced brain damage is through proteomic analysis. By the comparison of the proteome of neonatal brain cells exposed to HI with that of the mock-treated counterparts, differentially expressed and/or modified proteins could be identified. These proteins are potential markers of HI brain and they also provide a new foundation for further mechanistic analysis. In this study, we used two-dimensional gel electrophoresis and mass spectrometric (2D-MS) method to analyze neonatal mouse brain 24 h after HI and detected altered forms of collapsin response mediator proteins (CRMPs) in HI brain. The importance of CRMPs in neuronal function3–6 prompted us to determine the nature of this alteration and to further investigate the cause. We verified that CRMPs are hypophosphorylated in mouse brain after HI treatment. Moreover, a correlation of CRMP dephosphorylation with the downregulation of cyclin-dependent kinase 5 (CDK5) and CDK5 activator p35 was found. Our results had implications in the pathogenesis of HI-related neurological disorders.
Materials and Methods Animal Surgery. The induction of HI brain injury in 7-dayold C57/BL6N mice (3-4 g body weight) was conducted according to the Rice-Vannucci modification8 of the Levine Journal of Proteome Research 2008, 7, 2507–2515 2507 Published on Web 05/10/2008
research articles
Zhou et al.
9
method. This procedure has also been detailed in our previous publication.10 Briefly, mice were anesthetized with isoflurane (2.5% for induction and 1.5% for maintenance) and the right common carotid artery was isolated and doubly ligated with 5-0 surgical silk. Upon recovery, the mice were exposed to hypoxic gas (92% N2/8% O2) for 30 min. Subsequently, the mice were allowed to recover in the chambers under normoxic condition for 30 min, and then returned to their mothers. Animals were sacrificed 24 h after treatment and tissue samples were collected. Brains were dissected and sliced coronally into 1-mm sections. To confirm the area of damage, the sections were stained with triphenyltetrazolium chloride (TTC). Injured areas remained unstained with TTC, whereas healthy tissue was stained red. Brain damage was only evident in mice receiving both right common carotid artery ligation and hypoxia. Mice with artery ligation without hypoxia (L alone) or mice treated with hypoxia without ligation (H alone) were also included as controls in our experiments. Two-Dimensional Gel Electrophoresis and Mass Spectrometry. Two-dimensional (2D) electrophoresis, mass spectrometry and Western blotting were carried out as described.11 To prepare protein lysate, 200 mg of brain tissue from each mouse was cut into about 2 mm3 in size, lysed in 0.5 mL of Lysis Buffer (Bio-Rad) containing protease inhibitor cocktail (Sigma) and DNase I (1 unit/mL), and homogenized for 5 min on ice using a mini-homogenizer. To identify protein phosphorylation sites, matrix-assisted laser desorption/ionization (MALDI) was performed as previously described.11 High resolution spectra were acquired with Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems). Phosphorylation sites of peptides on Ser, Thr and Tyr residues can be determined by searching NCBInr protein database (http://prospector. ucsf.edu/) with MS-Fit. Peaks with observed masses that were higher by 80 Da or multiples of 80 Da than predicted were tentatively assigned as phosphopeptides. Western Blotting. Cell lysates containing 10 and 30 µg of protein were loaded onto SDS-PAGE and 2D-SDS-PAGE, respectively. Semidry transfer unit (model Hoefer TE77, GE Medical Systems) was used for electroblotting and protein bands were detected by ECL detection reagents (Amersham). Mouse monoclonal antibody C4G against human CRMP2 (486-528) and mouse monoclonal antibody 3F4 against hyperphosphorylated CRMP2 were kindly provided by Dr. Yasuo Ihara (Department of Neuropathology, Faculty of Medicine, University of Tokyo).12,13 Rabbit polyclonal antiserum C-19 against human p35, rabbit polyclonal antiserum C-8 against human CDK5, rabbit polyclonal anti-hypoxia-inducible factor 1R, and goat polyclonal anti-GRP78 were from Santa Cruz. Rabbit polyclonal anti-CRMP4 and mouse monoclonal antiCRMP5 antibodies were purchased from BD Biosciences. Mouse monoclonal antibody against R-tubulin (clone B5-1-2) was from Sigma. Protein Dephosphorylation Assay. Cell lysates containing 10 µg of protein were treated with 10 units of calf intestinal alkaline phosphatase (AP) for 1 h at 37 °C. The samples were then analyzed by SDS-PAGE followed by Western blotting. Immunoprecipitation and Protein Kinase Assay. The CDK5 kinase activity was determined as previously described.14,15 CDK5 was precipitated from brain lysate using anti-CDK5 antibody (Santa Cruz sc-173). Histone H1-derived peptide HS(1-18) with the sequence of KTPKKAKKPKTPKKAKKL was used as substrate for kinase assay with γ-[32P] ATP. The kinase reaction was performed for 30 min at 30 °C and a part of the 2508
Journal of Proteome Research • Vol. 7, No. 6, 2008
Figure 1. Western blot analysis of damage proteins in the mouse model of perinatal HI. Four groups of three mice were mocktreated (Control), were exposed to systemic hypoxia alone (H), underwent right common carotid artery ligation alone (L), or were treated with HI as a result of both H and L. The amounts of damage proteins (HIF-1R and GRP78) and control protein (Rtubulin) were analyzed by Western blotting.
reaction mixture (10 µL) was then spotted onto P81 paper (Whatman). Phosphate incorporation into substrate peptide was quantified using LS6500 liquid scintillation counter (Beckman).
Results Proteomic Analysis of HI Brain. To identify differentially expressed and/or modified proteins in HI brain, we used an established model of HI in 7-day-old mice,10 in which animals underwent unilateral common carotid artery ligation and were then exposed transiently to systemic hypoxia (8% O2). Consistent with our results of TTC staining (data not shown), the expression of damage proteins such as hypoxia-inducible factor 1R (HIF-1R) and GRP78 in the brain was significantly induced only in mice treated with HI, but not in mice receiving either right common artery ligation or systemic hypoxia alone (Figure 1). Thus, this model of perinatal HI is useful for our study. We then compared the proteomes of mock-treated and HItreated cortex and hippocampus by 2D gel electrophoresis. Representative gel images, with pI values ranging from 3 to 10 and molecular masses from 8 to 200 kDa, are presented in Figure 2A. More than 1500 spots were detected in each gel. The identity of 18 proteins from these 1500 spots was unanimously determined. Interestingly, among the proteins identified, a group of nine proteins was found to be significantly altered in both cortex and hippocampus 24 h after HI treatment. The changes were reproducibly seen in different mice (Figure 2B). MALDI-TOF-MS revealed that the nine proteins were CRMP2, CRMP4 and CRMP5 (Table 1). Several other proteins such as molecular chaperone HSC70, HSP60 and tubulin β5 were also identified (Table 1). Further analysis of the protein spots in cortex and hippocampus of four different mice confirmed that the relative levels of six protein spots representing CRMP2, CRMP4 and CRMP5 were elevated 2-19fold in HI brain, whereas the expression of three other forms of CRMP2 proteins was down-regulated 1.4-4.9-fold (Table 2). These multiple forms likely derived from CRMP proteins that are differentially modified post-translationally, raising the possibility that HI might specifically induce post-translational modification of CRMP proteins. Hypophosphorylated CRMPs in HI Brain. Since CRMPs are a family of neuron-enriched regulatory proteins important in neurite outgrowth2 and their activities are influenced by posttranslational modifications such as phosphorylation,12,16–20 we sought to investigate the alterations of CRMPs in HI brain using different methods. As a first step, we analyzed protein expres-
research articles
Hypophosphorylated CRMPs in Hypoxic-Ischemic Brain
Figure 2. Representative proteome profiles of cortex and hippocampus after HI. (A) Silver-stained 2D PAGE gels. Differentially expressed/ modified proteins are highlighted by arrows. Numbers indicate altered and excised protein spots. (B) Alterations of CRMP2, CRMP4 and CRMP5 in HI brain. CRMP protein spots on 2D gels are highlighted by arrows. Data are representative of three independent experiments. Table 1. Altered Expression of Proteins Identified by MALDI-TOF-MS spot no.
protein ID
MW/pI
NCBI accession no.
MOWSE score
675 674 768 856 950 949 127 137 682 301 302 35 280 921 219 932 809 888
collapsin response mediator protein 5 (CRMP-5) collapsin response mediator protein 5 (CRMP-5) collapsin response mediator protein 5 (CRMP-2) collapsin response mediator protein 5 (CRMP-4) collapsin response mediator protein 5 (CRMP-4) collapsin response mediator protein 5 (CRMP-4) collapsin response mediator protein 5 (CRMP-2) collapsin response mediator protein 5 (CRMP-2) collapsin response mediator protein 5 (CRMP-2) dnaK-type molecular chaperone HSC70 dnaK-type molecular chaperone HSC70 HSP 60 γ-actin creatine kinase ubiquinol-cytochrome C reductase complex core protein I tubulin β5 fascin homologue 1, Actin bundling protein unnamed protein product
61517/6.6 61517/6.6 62278/6.0 61937/6.0 61937/6.0 61937/6.0 62278/6.0 62278/6.0 62278/6.0 70838/5.4 70838/5.4 60956/5.9 41019/5.6 42714/5.4 52769/5.8 49671/4.8 54405/6.2 42185/6.6
12746424 M 12746424 M 40254595 M 6681219 M 6681219 M 6681219 M 40254595 M 40254595 M 40254595 M 476850 M 476850 M 3219998 M 809561 10946574 M 14548301 7106439 M 6679745 M 50815 M
2.96 × 105 6.89 × 103 3.73 × 108 1.12 × 106 3.58 × 109 3.10 × 1012 1.28 × 1013 2.84 × 1012 1.20 × 1012 1.81 × 1012 3.76 × 104 1.47 × 104 8.76 × 106 1.49 × 104 1.28 × 107 1.91 × 1012 3.87 × 107 1.81 × 104
sion using 1D and 2D Western blotting (Figure 3). The specificity of antibodies against CRMP2, CRMP4 and CRMP5 has previously been established.12,13,21 We also verified that these antibodies are highly specific and do not cross-react with different CRMP isoforms (data not shown). From 1D Western blot analysis (Figure 3A), fast-migrating protein bands reactive to anti-CRMP2, anti-CRMP4 and anti-CRMP5 antibodies were detected in HI-treated cortex and hippocampus from different
seq. coverage %
45 40 45 38 29 40 48 45 46 41 29 13 36 25 39 40 30 29
mice, but not in the untreated and unaffected control tissues on the contralateral hemisphere. Particularly, the fast-migrating CRMP2 band was evident in almost all samples of cortex and hippocampus, except for the hippocampus tissue of the F1 mouse, in which the fast-migrating band was weak. For CRMP4, the fast-migrating band was seen in three out of four mice. For CRMP5, while the fast migrating band could be visualized clearly in only one mouse, CRMP5-specific bands were not Journal of Proteome Research • Vol. 7, No. 6, 2008 2509
research articles
Zhou et al. a
Table 2. Altered Expression of Brain CRMPs in Four Mice Treated with HI cortex spots
protein ID
F1
hippocampus
F4
F5
674 675 768 856 949 950
CRMP5 CRMP5 CRMP2 CRMP4 CRMP4 CRMP4
3.9 9.4 3.0 1.6 2.6 1.0
6.5 23.7 8.4 8.2 3.0 3.0
4.9 25.0 3.6 5.6 2.1 2.7
127 137 682
CRMP2 CRMP2 CRMP2
2.0 1.7 1.8
1.8 1.3 1.9
1.7 1.6 1.4
M10
average
Up-Regulated 17.5 8.2 18.8 19.2 5.2 5.05 14.3 7.4 2.2 2.5 3.0 2.4 Down-Regulated 1.5 1.8 0.9 1.4 1.2 1.6
F1
F4
F5
M10
average
2.4 0.3 1.4 3.3 1.7 3.6
4.6 24.2 4.6 2.6 1.8 2.9
5.2 7.8 1.9 5.3 2.1 10.8
7.1 14.7 6.2 11.0 7.3 26.0
4.8 12.0 3.5 5.6 3.2 10.8
1.5 6.6 0.8
1.7 1.6 1.5
5.0 6.6 3.0
2.7 4.8 7.0
2.7 4.9 3.1
a Because brain damage was reproducibly seen in the ipsilateral but not the contralateral hemisphere, fold changes are calculated by comparing HI-treated cortex and hippocampus on the ipsilateral side with corresponding unaffected tissues on the contralateral side.
Figure 3. Western blot analysis of CRMP proteins. (A) 1D Western blot analysis. Total lysates of cortex and hippocampus from four mice (F1, F4, F5 and M10) treated with or without HI were subjected to Western blot analysis using antibodies against CRMP2, CRMP4 and CRMP5, respectively. The same blots were stripped and reprobed with anti-R-tubulin to verify equal loading. C: control. (B) Representative 2D Western blots. Blotting was preformed with cortex and hippocampus from mouse F5 only. Arrows point to CRMP protein spots. (C) Alkaline phosphatase treatment. Lysates of unaffected control cortex were treated with alkaline phosphatase (C+AP) prior to SDS-PAGE and Western analysis.
found in three other mice after HI (Figure 3A). While variations were observed in different mice, the fast-migrating bands were found only in HI mice. In agreement with the protein patterns in 1D Western blots, multiple fast-migrating protein spots reactive to anti-CRMP2, anti-CRMP4 and anti-CRMP5 antibodies were seen in 2D Western blots (Figure 3B), lending further support to the notion that CRMP proteins are differentially modified in HI brain. In 2510
Journal of Proteome Research • Vol. 7, No. 6, 2008
other words, the multiple fast-migrating spots detected in the 2D blots with higher resolution likely correspond to the fastmigrating bands seen in the lower-resolution 1D blots. On the other hand, the absence of CRMP protein bands in individual samples treated with HI, as seen with CRMP5 in the cortex of F1 mouse and in the cortex and hippocampus of M10 mouse, could be attributed to degradation or underexpression (Figure 3A).
research articles
Hypophosphorylated CRMPs in Hypoxic-Ischemic Brain The detection of fast-migrating protein species in 1D and 2D Western blots (Figure 3) is consistent with alteration in posttranslational modification of CRMPs. Because the regulatory roles of CRMP phosphorylation have been well-documented,12,16–20 we next investigated whether CRMP2, CRMP4 and CRMP5 were hypophosphorylated after HI treatment. For this purpose, we treated the protein samples with alkaline phosphatase (AP). Although the treatment of protein lysates from HI brain with AP did not alter the migration pattern of protein bands seen in Figure 3A, we could not rule out the possibility that the lysates of HI brain might contain an inhibitor of AP (data not shown). In contrast, treatment of protein lysates from unaffected control brain of the same animal led to the accumulation of a fast-migrating species (Figure 3C), which appeared at almost the same position as the fast-migrating band observed in Figure 3A. These results are compatible with the notion that the HI-induced fastmigrating CRMP proteins represent their hypophosphorylated forms.
It is generally accepted that the combination of unilateral carotid artery ligation with systemic hypoxia (L + H) produces cerebral ischemia and consequent brain damage in the ipsilateral but not the contralateral hemisphere.2 To verify that the dephosphorylation of CRMP proteins was specifically associated with HI-induced damage, but not the procedure of unilateral common carotid artery ligation (L alone) or the exposure to systemic hypoxia (H alone), we differentially treated mice with H alone, L alone, and L + H, and then examined the pattern of CRMPs by 1D and 2D Western blotting. Interestingly, dephosphorylation of CRMP2, CRMP4 and CRMP5 was not observed in mouse brain treated with either H or L alone (Figure 5). The fast-migrating hypophosphorylated CRMP bands (Figure 5A) and spots (Figure 5B) were detected on 1D and 2D gels, respectively, only after L + H treatment. Because similar results were obtained from three different mice and from all three forms of CRMPs, we were confident the dephosphorylation of CRMP proteins was specifically induced by HI as a result of L + H treatment.
Next, we further investigated the phosphorylation of CRMP2 using a previously characterized mouse monoclonal antibody (clone 3F4) specifically recognizing hyperphosphorylated CRMP2.12,13 When we probed the brain samples side by side with 3F4 antibody and another monoclonal antibody C4G reactive with total CRMP2 (Figure 4A), we noted that the slowmigrating CRMP2 species was recognized fairly well by both C4G and 3F4 antibodies. On the other hand, the fast-migrating CRMP2 species, which was seen in most HI samples except for the hippocampus of F13 and M19 mice, reacted strongly with C4G, but poorly with 3F4. That is to say, the slowmigrating species represent hyperphosphorylated CRMP2, whereas the fast-migrating species are hypophosphorylated. Thus, HI likely induced dephosphorylation of CRMP2 in mouse brain.
Dephosphorylation of CRMPs Is Associated with Inactivation of CDK5. CRMP proteins are known to be phosphorylated by several kinases including CDK5,18 GSK3,16,19,20 and Rho kinase.17 Among these CRMP kinases, CDK5 is a priming kinase, the action of which facilitates subsequent phosphorylation by other kinases.18,20 To investigate the cause of CRMP dephosphorylation after HI, we set out to determine the expression level of CDK5. CDK5 is a unique CDK which is activated not by cyclins, but by neuronal activator p35 or p39.15 p35 is therefore known as a regulatory subunit of CDK5 and can undergo further proteolytic activation by calpain to generate p25.22 Hence, we checked for the steady-state levels of both CDK5 and p35/p25 in HI brain (Figure 6A). While the amounts of CDK5 did not vary in control and HI brain, the levels of p35 were significantly lower in all samples of HI brain. Moreover, the relative amounts of p25 were also lower in some of these samples treated with HI (e.g., in mice F5, M10 and M6). This reduction was specifically induced by HI as a result of L + H treatment, but was not observed in the brain samples treated with H or L alone (Figure 6A, last four lanes on the right). Because down-regulation of p35/p25 might plausibly lead to inactivation of CDK5, we measured CDK5 activity in HI versus control brain using in vitro kinase assay and a synthetic substrate. Indeed, CDK5 activity recovered from HI brain by immunoprecipitation was significantly reduced compared to mock-treated brain (Figure 6B; P ) 0.02 by t test). Thus, inactivation of CDK5 might be causally associated with dephosphorylation of CRMP proteins after HI.
We repeated our experiments in a larger group of mice and quantitatively compared the relative amounts of hypophosphorylated CRMP2 versus total CRMP2 in control and HI brains. Generally consistent with our results shown in Figure 3 and Figure 4A, the ratios of hypophosphorylated CRMP2 were significantly higher in HI brain than in control brain (Figure 4B; P ) 0.023 by t test for cortex and P ) 0.0037 for hippocampus). To further confirm our findings, we also analyzed hyper- and hypophosphorylated forms of CRMPs using MALDI-TOF-MS. Two identical halves of a 2D gel were analyzed by Western blotting and silver staining. The protein spots on the silver-stained gel which correspond to spots reactive to anti-CRMP4 antibody on the immunoblot (Figure 4C) were excised and analyzed by MS (Figure 3B and Table 3). Comparison of the observed and the predicted masses of tryptic peptides verified the identity of the hyper- and hypophosphorylated forms of CRMP4 in HI brain. As such, form 1 of CRMP4 shown in Figure 4C had two phosphate groups at Thr-509 and Ser-522 (Figure 4D), whereas these phosphate groups were absent in form 6 of CRMP4. Thus, forms 1 and 6 represent hyper- and hypophosphorylated versions of CRMP4. Likewise, other hyper- and hypophosphorylated forms of CRMP2, CRMP4 and CRMP5 recovered from mock- and HI-treated brain were also confirmed by MALDI-TOF-MS. Notably, the phosphorylation sites identified in our study were generally consistent with those previously reported by other investigators.12,18–20 Collectively, our results indicated that HI induced dephosphorylation of CRMP2/4/5 at multiple sites.
Discussion In this study, we demonstrated the dephosphorylation of CRMP proteins in an animal model of perinatal brain damage induced by HI (Figure 1). This finding was supported by 1D and 2-D protein electrophoretic patterns (Figure 2), immunoblotting (Figure 3 and Figure 4B), treatment with alkaline phosphatase (Figure 3C), a phospho-specific antibody (Figure 4A), and MALDI-TOF-MS (Figure 4C,D; Tables 1–3). We also found that CRMP dephosphorylation occurred specifically after HI as a result of the combined action of unilateral common carotid artery ligation and systemic hypoxia (Figure 5). In addition, this dephosphorylation event might be caused by down-regulation of p35 leading to the inactivation of CDK5 kinase (Figure 6). Our work provided mechanistic insight into Journal of Proteome Research • Vol. 7, No. 6, 2008 2511
research articles
Zhou et al.
Figure 4. Analysis of hypophosphorylated CRMP2 and CRMP4 in HI brain by phospho-specific antibody and MS. (A) Western blot analysis of CRMP2 with anti-CRMP2 antibody (C4G) and anti-phospho-CRMP2 antibody (3F4). The same blot was stripped and reprobed with R-tubulin to normalize loading variation. C: control. (B) Quantitative analysis of hypophosphorylated CRMP2. The relative amounts of hypophosphorylated CRMP2 in control (C) and HI brain tissues were calculated. Results represent duplicate detection of paired C and HI samples from 12 mice. The error bars indicate standard deviation. The differences between the C and HI groups are statistically significant. R (%): percentages of hypophosphorylated CRMP2 in total CRMP2 (i.e., hypophosphorylated + hyperphosphorylated CRMP2). *: P ) 0.023 by t test. **: P ) 0.0037 by t test. (C and D) 2D Western blotting and 2D-MS. Equivalent 2D gels were analyzed by silver staining and Western blotting. Indicated protein spots were excised and analyzed by MS. Representative results are presented to demonstrate the identification of phosphorylation sites in CRMP4 based on MS spectrum. Protein samples were recovered from HItreated brain. Similar experiments were also conducted with CRMP2, CRMP4 and CRMP5 from control and HI-treated brain (data not shown).
the dephosphorylation of CRMPs in HI brain, which might be critically involved in HI-induced neurological diseases. CRMPs are a family of neuron-enriched, intracellular proteins and are expressed almost ubiquitously throughout the central nervous system.3,4 Their expression patterns are altered in an age-dependent manner.5 It is well-documented that CRMPs regulate neurite outgrowth and growth cone collapse through semaphorin3A (Sema3A), a repulsive guidance molecule.2 The expression of CRMPs is elevated during the peak period of axonal growth and diminished afterward.6 Among the five CRMP family members (CRMP1∼CRMP5) that are similar in structure and function, CRMP2 is the best studied isoform.7 2512
Journal of Proteome Research • Vol. 7, No. 6, 2008
CRMPs can impinge on many aspects of cellular function through interaction with partner proteins. For example, CRMP1/2 can interact with tubulin dimers to modulate microtubule dynamics and cytoskeletal organization.13,24 CRMPs are also involved in transducing intracellular signals induced by chondroitin sulfate proteoglycans,25,26 neurotrophins,27 Numb,28 reelin,29 and RhoA.26 However, the physiological functions of CRMPs are still largely unknown. Noteworthily, overexpression and underexpression of CRMPs have previously been shown to be associated with various neurological disease conditions, including impaired long-term potentiation,21 impaired spatial learning and memory,21 Alzheimer’s disease,18
research articles
Hypophosphorylated CRMPs in Hypoxic-Ischemic Brain Table 3. Phosphorylation of CRMP4 Detected by MALDI-TOF-MS spots
peptide submitteda
1
1257.5981 1721.7325 2207.0236 1257.6105 1721.7746 2207.5981 2041.9893 1257.6105 1721.7498 2041.9893 nd nd 1257.6105 1641.7834 1177.6442 1641.7834
2
3
4 5 6 a
modification
start-end
peptide sequence
possible phosphorylation sites
PO4 PO4 PO4 PO4 PO4 PO4 PO4 PO4 PO4 PO4
521-531 497-511 399-418 521-531 497-511 399-418 512-531 521-531 497-511 512-531
GSPTRPNPPVR GMYDGPVFDLTTTPK GRIAVGSDSDLVIWDPDALK GSPTRPNPPVR GMYDGPVFDLTTTPK GRIAVGSDSDLVIWDPDALK GGTPAGSTRGSPTRPNPPVR GSPTRPNPPVR GMYDGPVFDLTTTPK GGTPAGSTRGSPTRPNPPVR
S522, T524 Y499, T507-509 S405-407 S522, T524 Y499, T507-509 S405, S407 T514, S518, T519, S522, T524 S522, T524 Y499, T507-509 T514,S518,T519,S522,T524
PO4 No PO4 No PO4 No PO4
521-531 497-511 521-531 497-511
GSPTRPNPPVR GMYDGPVFDLTTTPK GSPTRPNPPVR GMYDGPVFDLTTTPK
S522, T524 Y499, T507-509 S522, T524 Y499, T507-509
Nd: non detectable.
dephosphorylation of CRMP might influence these processes. Particularly, calpain-mediated cleavage of CRMP2/3 and genetic knockout of CRMP1 have been demonstrated to induce neuronal apoptosis or degeneration;33,35,36 thus, it would be intriguing to find out whether hypophosphorylated CRMPs have proapoptotic activities. If dephosphorylation of CRMPs indeed triggers neuronal death, further studies should be conducted to shed mechanistic insight into this process and to explore the potential use of hypophosphorylated CRMPs as markers of brain damage.
Figure 5. Specific induction of CRMP dephosphorylation by HI. (A) Total lysates of cortex from 3 mice (M6, F12 and F13) were treated with H, L, and H + L as indicated in the box and analyzed by 1D Western blotting. C: control. (B) Representative 2D Western blot of protein lysate from M6 mouse treated as in panel A. Similar results were obtained from mice F12 and F13 (data not shown).
Our study was carried out in a well-established animal model of perinatal HI.2 The dephosphorylation of CRMPs was specifically observed after HI as a result of both unilateral common carotid artery ligation and systemic hypoxia (Figure 5). Either artery ligation or systemic hypoxia alone was insufficient to induce reduction of p35, activation of CDK5 or dephosphorylation of CRMPs (Figure 5 and Figure 6). This specificity of effect is explained by the fact that only a combination of artery ligation and systemic hypoxia can produce significant ischemia and brain damage.2 Interestingly, the dephosphorylation of CRMPs was observed only after 24 h post-HI, but not at earlier time points (data not shown). In addition, we failed to see rephosphorylation of CRMPs within 2-3 days post-HI (data not shown). Hence, CRMP dephosphorylation is a likely indicator of significant brain damage after transient HI.
binocular retinal lesions,30 neurotoxic and traumatic brain injury,31,32 as well as glutamate toxicity and cerebral ischemia.33,34 While our findings in the present study did not rule out the possibility that CRMPs are degraded in HI brain as previously shown in other models by other investigators,31–34 we provided the first evidence for HI-induced dephosphorylation of CRMPs. In addition, we demonstrated that dephosphorylation of CRMPs is specifically induced by HI (Figure 5). In this regard, it will be of interest to see whether dephosphorylation proceeds or even induces proteosome-dependent proteolysis of CRMPs. More importantly, further investigations are warranted to elucidate whether and how dephosphorylation of CRMPs mediates any of the severe and long-term sequelae in post-HI brain, particularly mental retardation and learning disability. Because CRMPs have been shown to have an impact on microtubule dynamics and apoptosis,13,24,33,35 additional experiments should be performed to clarify whether and how
We noted that some discrepancies in the detection of dephosphorylated CRMPs were found in different mice and different brain regions (Figure 3A and Figure 4A). While we cannot completely rule out the possibility that different CRMPs might behave differently in different brain regions, there was a generally consistent trend that dephosphorylated CRMPs were found in the brain only after HI. We have repeated our experiments in a larger group of mice (see Figure 4B for one example) and the results were highly reproducible. The data shown in this paper are representative of several rounds of experiments, in which a consistent trend of difference was not found. The variations observed were likely due to fluctuations in immunoassays and animal experiments. CRMP proteins can be phosphorylated by CDK5,18,37 GSK3,16,19,20 and Rho kinase.17,38 All these three groups of kinases serve important regulatory functions in neurons.39–42 Because the phosphorylation of CRMPs by CDK5 has a priming effect on the action of other kinases,18,20 it is not surprising Journal of Proteome Research • Vol. 7, No. 6, 2008 2513
research articles
Zhou et al.
Figure 6. HI-induced dephosphorylation of CRMPs was mediated through down-regulation of Cdk5/p35. (A) Protein lysates of cortex from different mice (F1, F4, F5, M10, M6, F12, F13 and M19) after HI treatment were probed with antibodies against p35/p25, CDK5, and R-tubulin. C: control. For comparison, a representative blot of samples treated with H alone, L alone, and HI as a result of L + H was also shown on the right. (B) CDK5 activity recovered from cortex after treatment with H alone, L alone and HI as a result of L + H for 24 h. CDK5 was immunoprecipitated from cortex and kinase activity was measured in vitro with histone H1 peptide as substrate. Results represent the average of percentage activity values of 6 mice. Statistically significant difference was found between groups C3 and HI (P ) 0.02 by t test).
that the dephosphorylation of CRMPs in HI brain correlates with down-regulation of CDK5 activity, plausibly caused by a reduction of p35 activator protein (Figure 6). These results support the notion that CDK5/p35 plays a crucial role in the regulation of CRMP activity.37 CDK5 is a neuronal kinase that phosphorylates many other substrates.43 However, the effects of CDK5 on growth cone collapse and microtubule dynamics are likely mediated by its phosphorylation and subsequent activation of CRMPs.18,37 In sharp contrast to the inactivation of CDK5, we found in our pilot experiments that GSK3 kinases are activated in HI brain (data not shown). However, it remains to be fully documented whether the other CRMP kinases are activated or inactivated in neonatal mouse brain after HI. Abbreviations: CRMP, collapsin response mediator protein; H, hypoxia; I, ischemia; L, ligation; 2D, two-dimensional; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ ionization; TOF, time-of-flight; 1D, one-dimensional; AP, alkaline phosphatase; GSK, glycogen synthase kinase; CDK, cyclin dependent kinase; Sema, Semaphorin.
Acknowledgment. We thank Dr. Yasuo Ihara for gifts of antibodies; Dr. Yick-Pang Ching for expert advice and reagent support; Dr. Dong-Yan Jin for useful discussion and assistance with manuscript preparation; and Dr. Andy Lau, Dr. Yick-Pang Ching and Dr. Amy Lo for critical reading of the manuscript. The study was funded by Hong Kong Research Grants Council (grant HKU7395/03M to J.-F.C.). References (1) Perlman, J. M. Summary proceedings from the neurology group on hypoxic-ischemic encephalopathy. Pediatrics 2006, 117, S28– S33. (2) Vannucci, R. C.; Vannucci, S. J. Perinatal hypoxic-ischemic brain damage: evolution of an animal model. Dev. Neurosci. 2005, 27, 81–86. (3) Quinn, C. C.; Gray, G. E.; Hockfield, S. A family of proteins implicated in axon guidance and outgrowth. J. Neurobiol. 1999, 41, 158–164.
2514
Journal of Proteome Research • Vol. 7, No. 6, 2008
(4) Charrier, E.; Reibel, S.; Rogemond, V.; Aguera, M.; Thomasset, N.; Honnorat, J. Collapsin response mediator proteins (CRMPs): involvement in nervous system development and adult neurodegenerative disorders. Mol. Neurobiol. 2003, 28, 51–64. (5) Cnops, L.; Hu, T. T.; Burnat, K.; Van der Gucht, E.; Arckens, L. Age-dependent alterations in CRMP2 and CRMP4 protein expression profiles in cat visual cortex. Brain Res. 2006, 1088, 109–119. (6) Bretin, S.; Reibel, S.; Charrier, E.; Maus-Moatti, M.; Auvergnon, N.; Thevenoux, A.; Glowinski, J.; Rogemond, V.; Premont, J.; Honnorat, J.; Gauchy, C. Differential expression of CRMP1, WTCRMP2A, WT-CRMP2B, and CRMP5 in axons or dendrites of distinct neurons in the mouse brain. J. Comp. Neurol. 2005, 486, 1–17. (7) Arimura, N.; Menager, C.; Fukata, Y.; Kaibuchi, K. Role of CRMP-2 in neuronal polarity. J. Neurobiol. 2004, 58, 34–47. (8) Rice, J. E.; Vannucci, R. C.; Brierley, J. B. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 1981, 9, 131–141. (9) Levine, S. Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 1960, 36, 1–17. (10) Tsang, M. C. S.; Lo, A. C. Y.; Cheung, P. T.; Chung, S. S. M.; Chung, S. K. Perinatal hypoxia-/ischemia-induced endothelin-1 mRNA in astrocyte-like and endothelial cells. NeuroReport 2001, 12, 2265– 2270. (11) Lau, A. T. Y.; Chiu, J.-F. The possible role of cytokeratin 8 in cadmium-induced adaptation and carcinogenesis. Cancer Res. 2007, 67, 2107–2113. (12) Gu, Y.; Hamajima, N.; Ihara, Y. Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522. Biochemistry 2000, 39, 4267–4275. (13) Gu, Y.; Ihara, Y. Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules. J. Biol. Chem. 2000, 275, 17917–1792. (14) Lew, J.; Beaudette, K.; Litwin, C. M. E.; Wang, J. H. Brain prolinedirected protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2. J. Biol. Chem. 1992, 267, 13383–13390. (15) Ching, Y. P.; Pang, A. S.; Lam, W. H.; Qi, R. Z.; Wang, J. H. Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor. J. Biol. Chem. 2002, 277, 15237–15240. (16) Cole, A. R.; Knebel, A.; Morrice, N. A.; Robertson, L. A.; Irving, A. J.; Connolly, C. N.; Sutherland, C. GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons. J. Biol. Chem. 2004, 279, 50176–50180.
research articles
Hypophosphorylated CRMPs in Hypoxic-Ischemic Brain (17) Arimura, N.; Menager, C.; Kawano, Y.; Yoshimura, T.; Kawabata, S.; Hattori, A.; Fukata, Y.; Amano, M.; Goshima, Y.; Inagaki, M.; Morone, N.; Usukura, J.; Kaibuchi, K. Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones. Mol. Cell. Biol. 2005, 25, 9973–9984. (18) Uchida, Y.; Ohshima, T.; Sasaki, Y.; Suzuki, H.; Yanai, S.; Yamashita, N.; Nakamura, F.; Takei, K.; Ihara, Y.; Mikoshiba, K.; Kolattukudy, P.; Honnorat, J.; Goshima, Y. Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3β phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer’s disease. Genes Cells 2005, 10, 165– 179. (19) Yoshimura, T.; Kawano, Y.; Arimura, N.; Kawabata, S.; Kikuchi, A.; Kaibuchi, K. GSK-3β regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 2005, 120, 137–149. (20) Cole, A. R.; Causeret, F.; Yaeirgi, G.; Hastie, C. J.; McLauchlan, H.; McManus, E. J.; Hernandez, F.; Eickholt, B. J.; Nikolic, M.; Sutherland, C. Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo. J. Biol. Chem. 2006, 281, 16591–16598. (21) Su, K.-Y.; Chien, W.-L.; Fu, W.-M.; Yu, I.-S.; Huang, H.-P.; Huang, P.-H.; Lin, S.-R.; Shih, J.-Y.; Lin, Y.-L.; Hsueh, Y.-P.; Yang, P.-C.; Lin, S.-W. Mice deficient in collapsin response mediator protein-1 exhibit impaired long-term potentiation and impaired spatial learning and memory. J. Neurosci. 2007, 27, 2513–2524. (22) Lew, J.; Huang, Q. Q.; Qi, Z.; Winkfein, R. J.; Aebersold, R.; Hunt, T.; Wang, J. H. A brain-specific activator of cyclin-dependent kinase 5. Nature 1994, 371, 423–426. (23) Goshima, Y.; Nakamura, F.; Strittmatter, P.; Strittmatter, S. M. Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33. Nature 1995, 376, 509–514. (24) Fukata, Y.; Itoh, T. J.; Kimura, T.; Menager, C.; Nishimura, T.; Shiromizu, T.; Watanabe, H.; Inagaki, N.; Iwamatsu, A.; Hotani, H.; Kaibuchi, K. CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat. Cell Biol. 2002, 4, 583–591. (25) Franken, S.; Junghans, U.; Rosslenbroich, V.; Baader, S. L.; Hoffmann, R.; Gieselmann, V.; Viebahn, C.; Kappler, J. Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate. J. Biol. Chem. 2003, 278, 3241–3250. (26) Alabed, Y. Z.; Pool, M.; Tone, S. O.; Fournier, A. E. Identification of CRMP4 as a convergent regulator of axon outgrowth inhibition. J. Neurosci. 2007, 27, 1702–1711. (27) Quach, T. T.; Duchemin, A. M.; Rogemond, V.; Aguera, M.; Honnorat, J.; Belin, M. F.; Kolattukudy, P. E. Involvement of collapsin response mediator proteins in the neurite extension induced by neurotrophins in dorsal root ganglion neurons. Mol. Cell. Neurosci. 2004, 25, 433–443. (28) Nishimura, T.; Fukata, Y.; Kato, K.; Yamaguchi, T.; Matsuura, Y.; Kamiguchi, H.; Kaibuchi, K. CRMP-2 regulates polarized Numbmediated endocytosis for axon growth. Nat. Cell Biol. 2003, 5, 819– 826. (29) Yamashita, N.; Uchida, Y.; Ohshima, T.; Hirai, S.; Nakamura, F.; Taniguchi, M.; Mikoshiba, K.; Honnorat, J.; Kolattukudy, P.; Thomasset, N.; Takei, K.; Takahashi, T.; Goshima, Y. Collapsin
(30)
(31)
(32)
(33)
(34)
(35)
(36) (37)
(38)
(39) (40) (41) (42) (43)
response mediator protein 1 mediates reelin signaling in cortical neuronal migration. J. Neurosci. 2006, 26, 13357–13362. Cnops, L.; Hu, T. T.; Eysel, U. T.; Arckens, L. Effect of binocular retinal lesions on CRMP2 and CRMP4 but not Dyn I and Syt I expression in adult cat area 17. Eur. J. Neurosci. 2007, 25, 1395– 1401. Zhang, Z.; Ottens, A. K.; Sadasivan, S.; Kobeissy, F. H.; Fang, T.; Hayes, R. L.; Wang, K. K. W. Calpain-mediated collapsin response mediator protein-1,-2, and-4 proteolysis after neurotoxic and traumatic brain injury. J. Neurotrauma 2007, 24, 460–472. Kobeissy, F. H.; Ottens, A. K.; Zhang, Z.; Liu, M. C.; Denslow, N. D.; Dave, J. R.; Tortella, F. C.; Hayes, R. L.; Wang, K. K. W. Novel differential neuroproteomics analysis of traumatic brain injury in rats. Mol. Cell. Proteomics 2006, 5, 1887–1898. Hou, S. T.; Jiang, S. X.; Desbois, A.; Huang, D.; Kelly, J.; Tessier, L.; Karchewski, L.; Kappler, J. Calpain-cleaved collapsin response mediator protein-3 induces neuronal death after glutamate toxicity and cerebral ischemia. J. Neurosci. 2006, 26, 2241–2249. Hu, X.; Rea, H. C.; Wiktorowicz, J. E.; Perez-Polo, J. R. Proteomic analysis of hypoxia/ischemia-induced alteration of cortical development and dopamine neurotransmission in neonatal rat. J. Proteome Res. 2006, 5, 2396–2404. Charrier, E.; Mosinger, B.; Meissirel, C.; Aguera, M.; Rogemond, V.; Reibel, S.; Salin, P.; Chounlamountri, N.; Perrot, V.; Belin, M. F.; Goshima, Y.; Honnorat, J.; Thomasset, N.; Kolattukudy, P. Transient alterations in granule cell proliferation, apoptosis and migration in postnatal developing cerebellum of CRMP1-/-mice. Genes Cells 2006, 11, 1337–1352. Touma, E.; Kato, S.; Fukui, K.; Koike, T. Calpain-mediated cleavage of collapsin response mediator protein(CRMP)-2 during neurite degeneration in mice. Eur. J. Neurosci. 2007, 26, 3368–3381. Brown, M.; Jacobs, T.; Eickholt, B.; Ferrari, G.; Teo, M.; Monfries, C.; Qi, R. Z.; Leung, T.; Lim, L.; Hall, C. R2-chimaerin, cyclindependent kinase 5/p35, and its target collapsin response mediator protein-2 are essential components in semaphorin 3A-induced growth-cone collapse. J. Neurosci. 2004, 24, 8994–9004. Hall, C.; Brown, M.; Jacobs, T.; Ferrari, G.; Cann, N.; Teo, M.; Monfries, C.; Lim, L. Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase. J. Biol. Chem. 2001, 276, 43482–43486. Cruz, J. C.; Tsai, L. H. Cdk5 deregulation in the pathogenesis of Alzheimer’s disease. Trends Mol. Med. 2004, 10, 452–458. Cheung, Z. H.; Fu, A. K.; Ip, N. Y. Synaptic roles of Cdk5: implications in higher cognitive functions and neurodegenerative diseases. Neuron 2006, 50, 13–18. Frame, S.; Cohen, P. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 2001, 359, 1–16. Mueller, B. K.; Mack, H.; Teusch, N. Rho kinase, a promising drug target for neurological disorders. Nat. Rev. Drug Discovery 2005, 4, 387–398. Horiuchi, Y.; Asada, A.; Hisanaga, S.-J.; Toh-e, A.; Nishizawa, M. Identifying novel substrates for mouse Cdk5 kinase using the yeast Saccharomyces cerevisiae. Genes Cells 2006, 11, 1393–1404.
PR800108K
Journal of Proteome Research • Vol. 7, No. 6, 2008 2515
