Research ArticlePhosphorylation of CRMP2 by Cdk5 Regulates Dendritic SpineDevelopment of Cortical Neuron in the Mouse Hippocampus

Xiaohua Jin1 Kodai Sasamoto1 Jun Nagai1 Yuki Yamazaki1 Kenta Saito2

Yoshio Goshima3 Takafumi Inoue2 and Toshio Ohshima1

1Laboratory for Molecular Brain Science Department of Life Science and Medical Bioscience Waseda UniversityTokyo 162-8480 Japan2Laboratory for Neurophysiology Department of Life Science and Medical Bioscience Waseda University Tokyo 162-8480 Japan3Department of Molecular Pharmacology and Neurobiology Graduate School of Medicine Yokohama City UniversityYokohama 236-0004 Japan

Correspondence should be addressed to Toshio Ohshima ohshimawasedajp

Received 7 June 2015 Revised 11 July 2015 Accepted 21 July 2015

Academic Editor Kwok-On Lai

Copyright copy 2016 Xiaohua Jin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memoryHowever our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines islimited We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendriticspines of cortical neurons in the mouse brain Previous in vitro studies have suggested the involvement of Cdk5 substrates in theformation of dendritic spines however their role in spine development has not been tested in vivo Here we demonstrate that Cdk5phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons and invivo in the mouse brain When we eliminated CRMP2 phosphorylation in CRMP2KIKI mice the densities of dendritic spinessignificantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain These results indicate that phosphorylation ofCRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus

1 Introduction

For the development of functional neural circuitry the for-mation of synapses between appropriate partners is a criticalstepThemajority of excitatory synapses of postsynaptic neu-rons are localized in specialized cellular structures called den-dritic spines The formation maturation and maintenanceof dendritic spines are tightly regulated by different extracel-lular signals including semaphorin 3A (Sema3A) Collapsinresponse mediator proteins (CRMPs) initially identified asa signaling molecule of Sema3A [1] are composed of fivehomologous cytosolic phosphoproteins (CRMP1ndash5) and arehighly expressed in developing and adult nervous systems[2ndash5] CRMPs bind with tubulin heterodimers whereas thesequential phosphorylation of CRMPs lowers their bindingaffinity to tubulin [6] CRMP2 also colocalizes with the actincytoskeleton [7] and coimmunoprecipitates with actin [8 9]

Phosphorylation of CRMP1 and CRMP2 by Cdk5 andsequential phosphorylation of CRMP2 by GSK-3120573 are crucialfor Sema3A-induced growth cone collapse response in dorsalroot ganglia (DRG) neurons [10 11]

Recently we demonstrated that Cdk5p35 is necessaryfor dendritic spine development and maintenance [12]Additionally we previously showed that Sema3A-inducedspine development is mediated through phosphorylationof CRMP1 by Cdk5 [13] and that CRMP1 and CRMP2have functional redundancy in neuronal development [14]Therefore we hypothesized that phosphorylation of CRMP2by Cdk5 is also important for the development of dendriticspines in vivo To test this we first analyzed the localizationof phosphorylated forms of CRMP2 in the synapses ofcultured hippocampal neurons and in vivo in the mousehippocampus We observed phosphorylation of CRMP2 byCdk5 in the dendritic spines of hippocampal neurons in vitro

Hindawi Publishing CorporationNeural PlasticityVolume 2016 Article ID 6790743 7 pageshttpdxdoiorg10115520166790743

2 Neural Plasticity

and in vivo We then analyzed spine densities of hippocampalCA1 pyramidal neurons in CRMP2KIKI mice in which theCdk5 phosphorylation site of CRMP2 at amino acid 522 waschanged from Ser to Ala [14] We found reduced dendriticspine densities in hippocampal neurons in CRMP2KIKI miceThese results indicate that CRMP2 phosphorylation by Cdk5is important for the development of dendritic spines inhippocampal neurons in vivo

2 Materials and Methods

21 Mice The mice used in our experiments were housedin accordance with protocols approved by the Institu-tional Animal Care and Use Committee at Waseda Uni-versity CRMP2KIKI mice were generated and genotyped asdescribed previously [14] GFP-M mice a gift from J Sanes[15] were crossed with these mutant mice for the presentstudy

22 Neuronal Culture and Immunocytochemistry Primarycultures of hippocampal neurons were prepared from E18Wistar rats as described previously [16] with the follow-ing modifications cells were plated at a density of 50 times104 cellswell on coverslips coated with 200120583gmL poly-L-lysine (Sigma Japan Tokyo) in 24-well plates Neurobasal-A (Life Technologies Japan Tokyo) B27-supplement (Mil-tenyi Biotec Tokyo) 2mM L-glutamine (Life TechnologiesJapan) and penicillinstreptomycin (Nacalai Tesque Kyoto)were used as culture medium Immunocytochemistry wasperformed as previously described [17] Briefly after washingwith phosphate-buffered saline (PBS) cells were fixed with4 paraformaldehyde (PFA) for 15min at room temperature(RT) After washing with PBS cells were incubated withprimary antibodies which were diluted in PBS001 TritonX-100 at 4∘C overnight They were then washed 3 timeswith PBS and incubated with Alexa-Fluor 488 (1 1000) orAlexa-Fluor 568 (1 1000) secondary antibodies for 1 h After3 further washes with PBS the sections were embeddedin Vectashield mounting media (Vector Labs BurlingameCA) Images were obtained using a laser scanning confocalmicroscope based on an FV1000 scanning unit (OlympusJapan) Primary antibodies used in this study are anti-PSD95 (mouse monoclonal Millipore) anti-synaptophysin(mouse monoclonal Millipore) and pCRMP2(S522) whichrecognizes phospho-CRMP2 at Ser522 (rabbit polyclonalEMC Biosciences)

23 Histological Analysis

231 Immunohistochemistry Mice were anesthetized usingdiethyl ether and then perfused transcardially with 4 PFAin PBS Brain samples were fixed in 4 PFA in PBS overnightat 4∘C GFP-M mice used in this study were 4ndash6 weeksof age After dehydration in 20 sucrose in PBS sampleswere embedded in OCT compound (Sakura Finetek Japan)Cryosections were cut at 14 120583m thickness For immunos-taining sections were incubated with anti-pCRMP2(S522)antibody at 4∘C overnight After washing with PBS thesecondary antibody AlexaFluor was applied and sections

were mounted with Vectashield All immunostaining imageswere captured with a confocal microscope (FV1000)

232 RapidGolgi Staining Male CRMP2KIKI andCRMP2++mice at P18 and at 5 weeks of age (119899 = 3 for each genotype andage)were used in this study FormodifiedGolgi-Cox stainingan FDRapidGolgiStain kitwas used (FDNeuroTechnologiesMD) Stained slices were sectioned at a thickness of 200120583mPyramidal hippocampal CA1 neurons in each mouse wereselected for the analysis as described in our previouswork [1213] Dendritic spines of CA1 pyramidal neurons were countedin 50 120583m segments of proximal branches of apical dendritesunder a BX50 microscope (Olympus) with a UPlanSApo40x (NA = 095) objective In a typical experiment morethan 2000 spines were counted on more than 50 dendriticsegments in 25 neurons Average spine densities per 50120583mdendritic segments were then calculated for each genotypegroup Groups of spines were compared using Studentrsquos 119905-test

3 Results

31 CRMP2 Is Phosphorylated in Dendritic Spines of CulturedHippocampal Neurons We tested the possible function ofCRMP2 phosphorylation in synapses We first examined thesubcellular localization of phospho-CRMP2 (pCRMP2) incultured hippocampal neurons We used anti-synaptophysinor anti-PSD-95 antibodies as presynaptic and postsynapticmarkers respectively Double staining with anti-pCRMP2and anti-synaptophysin or anti-PSD-95 antibodies showedthat pCRMP2 colocalized with both synaptophysin andPSD-95 in dendritic protrusions (Figure 1) These resultsdemonstrate that CRMP2 is phosphorylated by Cdk5 in thepresynapse and dendritic spines in cultured hippocampalneurons suggesting the possible involvement of CRMP2phosphorylation in the development of dendritic spines inhippocampal neurons

32 Reduced Spine Densities of Hippocampal CA1 PyramidalNeurons in Juvenile CRMP2119870119868119870119868 Mice We examined den-dritic spine density by Golgi staining in P18 CRMP2KIKImice Golgi staining of forebrain slices showed a reduction inthe number of spines in hippocampal CA1 pyramidal neuronsinCRMP2KIKImice comparedwith those inCRMP2++mice(Figure 2) These results indicate that phosphorylation ofCRMP2byCdk5 is required for proper formation of dendriticspines in the mouse brain

33 CRMP2 Is Phosphorylated in Dendritic Spines of Hip-pocampal CA1 Pyramidal Neurons in Mouse Brains CRMP2is expressed in hippocampal neurons in adult mice [2]Thus we examined its phosphorylation in dendritic spinesin hippocampal CA1 pyramidal neurons For this purposewe performed immunostaining of hippocampal sectionsfrom GFP-M mice at 4ndash6 weeks of age with anti-pCRMP2antibody In GFP-M mice some hippocampal CA1 pyra-midal neurons express GFP [15] As shown in Figure 3 wedetected pCRMP2 immunoreactivity in dendritic spines inhippocampal CA1 pyramidal neurons of GFP-M mice In

Neural Plasticity 3

SynaptophysinpCRMP2S522

PSD95pCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 1 Subcellular localization of phospho-CRMP2 in cultured hippocampal neurons (a) Immunocytochemistry with anti-phospho-CRMP2 and synaptophysin antibodies Higher magnification is shown in (a1015840) (b) Immunocytochemistry with anti-phospho-CRMP2 andPSD95 antibodies in cultured rat hippocampal neurons 28 days in vitro (DIV) Merged images are shown Higher magnification shown in(b1015840) Scale bar 20 120583m

contrast its immunoreactivity was very low in those of GFP-M CRMP2KIKI double mutant which is attributable to across reactivity of this antibody to pCRMP1 [14]These resultssuggest that Cdk5 phosphorylates CRMP2 in dendritic spinesof hippocampal CA1 pyramidal neurons in the mouse brain

34 Reduced Spine Densities of Hippocampal CA1 PyramidalNeurons in 5-Week-Old CRMP2119870119868119870119868 Mice We examineddendritic spine density by Golgi staining in 5-week-oldCRMP2KIKI mice Golgi staining of forebrain slices showeda reduction in the numbers of spines in hippocampal CA1pyramidal neurons in CRMP2KIKI mice compared withthose in CRMP2++ mice (Figure 4) These results indicatethat phosphorylation of CRMP2 by Cdk5 is required for thedevelopment of proper dendritic spine density in the adultmouse brain

4 Discussion

Recent studies have demonstrated that Cdk5 substrates areinvolved in the regulation of spine formation Synapticproteins phosphorylated by Cdk5 including ephexin1 [18]WAVE1 [19] CRMP1 [13] TrkB [20] PSD95 [21] drebrin[22] and p70 ribosomal S6 kinase (S6K) [23] have beenshown to play a role in spine formation [13 19 21 23]and maintenance [18 20] However their functions differsuch that some of them are crucial for spine formation [13]and some for spine retraction [18] We recently reportedreductions of dendritic spine densities in hippocampal CA1pyramidal neurons of inducible-p35 cKO p39 KO mice andCA1-p35 cKO p39 KO mice with a p35 deletion in the CA1region of the hippocampus after P17 [12] We also reportedreduction of spine densities in cerebral layer V neurons andhippocampal CA1 pyramidal neurons in inducible-p35 cKO

4 Neural Plasticity

CRMP2++

CRMP2KIKI

(a)

lowastlowast

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 2 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in CRMP2KIKI mice at P18 (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at P18 Scale bar 10 120583m (b) Reduced dendritic spine densitywas observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in each area fromthree mice in each genotype were analyzed lowast119875 lt 005

GFPpCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 3 Localization of phospho-CRMP2 at dendritic spines of hippocampal CA1 pyramidal neurons (a) Representative images ofimmunostaining of apical dendrites and their branches with phospho-CRMP2(S522) (pCRMP2S522) antibody in hippocampal CA1pyramidal neurons from GFP-M mice Magnified images of the areas indicated in (a) are shown in (a1015840) Scale bar 10 120583m (b) Representativeimages of immunostaining of apical dendrites and their branches with pCRMP2S522 antibody of hippocampal CA1 pyramidal neurons inGFP-M CRMP2KIKI mice Magnified images of the areas indicated in (b) are shown in (b1015840) Scale bar 10 120583m

Neural Plasticity 5

CRMP2++

CRMP2KIKI

(a)

lowastlowast

45

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 4 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in 5 week-old CRMP2KIKI mice (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at 5 weeks old Scale bar 10 120583m (b) Reduced dendritic spinedensity was observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in eacharea from three mice in each genotype were analyzed lowastlowast119875 lt 001

p39 KO mice when we deleted the p35 gene in 4-month-oldanimals [12]These findings indicate that spine formation andmaintenance are dependent on Cdk5 kinase activity in themouse brain

Our previous study showed that Sema3A-induced spinedevelopment is mediated by phosphorylation of CRMP1 byCdk5 [13] Because CRMP1 and CRMP2 have functionalsimilarities in brain development [14] we examined whetherphosphorylation of CRMP2 by Cdk5 is also important forthe development andmaintenance of dendritic spines in vivoCdk5 specifically phosphorylates Ser residue of CRMP2 at522 [10] We previously generated CRMP2KIKI mice to studythe function of Cdk5-mediated CRMP2 phosphorylationby replacing Ser at 522 to Ala [14] Our present analysisof dendritic spine densities in hippocampal CA1 pyramidalneurons in CRMP2KIKI mice at P18 showed reduced spinedensities in these neurons compared to those of controls(Figure 2) Along with our previous study on CRMP1KOmice [13] these results indicate that CRMPs are importantsubstrates of Cdk5 for spine formation The results obtainedin the present study will provide a new insight into theregulatory mechanisms underlying the effect of Cdk5 ondendritic spine density

Our analysis of 5-week-old CRMP2KIKI mice showedfurther reduction of spine densities in hippocampal CA1pyramidal neurons (Figure 4) These results exclude the pos-sibility that reduced spine densities of hippocampal neuronsin CRMP2KIKI mice at P18 (Figure 2) are due to the delayof brain development In the cerebral cortex of macaquemonkeys and humans the number of dendritic spines rapidly

increases after birth and peaks in an early phase of theinfantile period [24] Spine density then decreases duringthe later infantile period and adolescence period to reachthe adult level [25] Decrease of dendritic spine densityduring the transition from puberty to adulthood has alsobeen reported in the mouse hippocampus [26]These studiesindicate ontogenetic similarity between rodent primate andhuman in spine formation and pruning This overshoot-typetime course of spine formation and pruning is attractivefor researchers because it is possibly involved in develop-mental and psychiatric disorders [27] Further studies arealso required for the analysis of the involvement of CRMP2phosphorylation in spine pruning and maintenance

Cdk5 and its activator p35 play multiple roles in braindevelopment especially in neuronal migration [28] Emerg-ing evidence suggests that Cdk5p35 is also involved insynaptic plasticity [29] Cdk5p35 is localized at neuronalsynapses and phosphorylates many synaptic proteins [21 30ndash33] Furthermore the induction of synaptic plasticity andspatial learning are impaired in Cdk5p35 mutant mice [34ndash36] The role of Cdk5 in synaptic plasticity and learning wasinitially studied using Cdk5 inhibitors which showed inhibi-tion of hippocampal LTP induction and context-dependentfear conditioning [30 37] We have previously reported theimpairment of long-term depression (LTD) induction andspatial learning and memory in p35 KO mice [36] Ourrecent study of p35 conditional KO (cKO) mice which lackhistological abnormalities in the brain also showed impair-ment of spatial learning and memory and LTD induction[38] Importantly electrophysiological analysis of hippocam-pal slices from p35 cKO mice revealed reduced synaptic

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

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2 Neural Plasticity

and in vivo We then analyzed spine densities of hippocampalCA1 pyramidal neurons in CRMP2KIKI mice in which theCdk5 phosphorylation site of CRMP2 at amino acid 522 waschanged from Ser to Ala [14] We found reduced dendriticspine densities in hippocampal neurons in CRMP2KIKI miceThese results indicate that CRMP2 phosphorylation by Cdk5is important for the development of dendritic spines inhippocampal neurons in vivo

2 Materials and Methods

21 Mice The mice used in our experiments were housedin accordance with protocols approved by the Institu-tional Animal Care and Use Committee at Waseda Uni-versity CRMP2KIKI mice were generated and genotyped asdescribed previously [14] GFP-M mice a gift from J Sanes[15] were crossed with these mutant mice for the presentstudy

22 Neuronal Culture and Immunocytochemistry Primarycultures of hippocampal neurons were prepared from E18Wistar rats as described previously [16] with the follow-ing modifications cells were plated at a density of 50 times104 cellswell on coverslips coated with 200120583gmL poly-L-lysine (Sigma Japan Tokyo) in 24-well plates Neurobasal-A (Life Technologies Japan Tokyo) B27-supplement (Mil-tenyi Biotec Tokyo) 2mM L-glutamine (Life TechnologiesJapan) and penicillinstreptomycin (Nacalai Tesque Kyoto)were used as culture medium Immunocytochemistry wasperformed as previously described [17] Briefly after washingwith phosphate-buffered saline (PBS) cells were fixed with4 paraformaldehyde (PFA) for 15min at room temperature(RT) After washing with PBS cells were incubated withprimary antibodies which were diluted in PBS001 TritonX-100 at 4∘C overnight They were then washed 3 timeswith PBS and incubated with Alexa-Fluor 488 (1 1000) orAlexa-Fluor 568 (1 1000) secondary antibodies for 1 h After3 further washes with PBS the sections were embeddedin Vectashield mounting media (Vector Labs BurlingameCA) Images were obtained using a laser scanning confocalmicroscope based on an FV1000 scanning unit (OlympusJapan) Primary antibodies used in this study are anti-PSD95 (mouse monoclonal Millipore) anti-synaptophysin(mouse monoclonal Millipore) and pCRMP2(S522) whichrecognizes phospho-CRMP2 at Ser522 (rabbit polyclonalEMC Biosciences)

23 Histological Analysis

231 Immunohistochemistry Mice were anesthetized usingdiethyl ether and then perfused transcardially with 4 PFAin PBS Brain samples were fixed in 4 PFA in PBS overnightat 4∘C GFP-M mice used in this study were 4ndash6 weeksof age After dehydration in 20 sucrose in PBS sampleswere embedded in OCT compound (Sakura Finetek Japan)Cryosections were cut at 14 120583m thickness For immunos-taining sections were incubated with anti-pCRMP2(S522)antibody at 4∘C overnight After washing with PBS thesecondary antibody AlexaFluor was applied and sections

were mounted with Vectashield All immunostaining imageswere captured with a confocal microscope (FV1000)

232 RapidGolgi Staining Male CRMP2KIKI andCRMP2++mice at P18 and at 5 weeks of age (119899 = 3 for each genotype andage)were used in this study FormodifiedGolgi-Cox stainingan FDRapidGolgiStain kitwas used (FDNeuroTechnologiesMD) Stained slices were sectioned at a thickness of 200120583mPyramidal hippocampal CA1 neurons in each mouse wereselected for the analysis as described in our previouswork [1213] Dendritic spines of CA1 pyramidal neurons were countedin 50 120583m segments of proximal branches of apical dendritesunder a BX50 microscope (Olympus) with a UPlanSApo40x (NA = 095) objective In a typical experiment morethan 2000 spines were counted on more than 50 dendriticsegments in 25 neurons Average spine densities per 50120583mdendritic segments were then calculated for each genotypegroup Groups of spines were compared using Studentrsquos 119905-test

3 Results

31 CRMP2 Is Phosphorylated in Dendritic Spines of CulturedHippocampal Neurons We tested the possible function ofCRMP2 phosphorylation in synapses We first examined thesubcellular localization of phospho-CRMP2 (pCRMP2) incultured hippocampal neurons We used anti-synaptophysinor anti-PSD-95 antibodies as presynaptic and postsynapticmarkers respectively Double staining with anti-pCRMP2and anti-synaptophysin or anti-PSD-95 antibodies showedthat pCRMP2 colocalized with both synaptophysin andPSD-95 in dendritic protrusions (Figure 1) These resultsdemonstrate that CRMP2 is phosphorylated by Cdk5 in thepresynapse and dendritic spines in cultured hippocampalneurons suggesting the possible involvement of CRMP2phosphorylation in the development of dendritic spines inhippocampal neurons

32 Reduced Spine Densities of Hippocampal CA1 PyramidalNeurons in Juvenile CRMP2119870119868119870119868 Mice We examined den-dritic spine density by Golgi staining in P18 CRMP2KIKImice Golgi staining of forebrain slices showed a reduction inthe number of spines in hippocampal CA1 pyramidal neuronsinCRMP2KIKImice comparedwith those inCRMP2++mice(Figure 2) These results indicate that phosphorylation ofCRMP2byCdk5 is required for proper formation of dendriticspines in the mouse brain

33 CRMP2 Is Phosphorylated in Dendritic Spines of Hip-pocampal CA1 Pyramidal Neurons in Mouse Brains CRMP2is expressed in hippocampal neurons in adult mice [2]Thus we examined its phosphorylation in dendritic spinesin hippocampal CA1 pyramidal neurons For this purposewe performed immunostaining of hippocampal sectionsfrom GFP-M mice at 4ndash6 weeks of age with anti-pCRMP2antibody In GFP-M mice some hippocampal CA1 pyra-midal neurons express GFP [15] As shown in Figure 3 wedetected pCRMP2 immunoreactivity in dendritic spines inhippocampal CA1 pyramidal neurons of GFP-M mice In

Neural Plasticity 3

SynaptophysinpCRMP2S522

PSD95pCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 1 Subcellular localization of phospho-CRMP2 in cultured hippocampal neurons (a) Immunocytochemistry with anti-phospho-CRMP2 and synaptophysin antibodies Higher magnification is shown in (a1015840) (b) Immunocytochemistry with anti-phospho-CRMP2 andPSD95 antibodies in cultured rat hippocampal neurons 28 days in vitro (DIV) Merged images are shown Higher magnification shown in(b1015840) Scale bar 20 120583m

contrast its immunoreactivity was very low in those of GFP-M CRMP2KIKI double mutant which is attributable to across reactivity of this antibody to pCRMP1 [14]These resultssuggest that Cdk5 phosphorylates CRMP2 in dendritic spinesof hippocampal CA1 pyramidal neurons in the mouse brain

34 Reduced Spine Densities of Hippocampal CA1 PyramidalNeurons in 5-Week-Old CRMP2119870119868119870119868 Mice We examineddendritic spine density by Golgi staining in 5-week-oldCRMP2KIKI mice Golgi staining of forebrain slices showeda reduction in the numbers of spines in hippocampal CA1pyramidal neurons in CRMP2KIKI mice compared withthose in CRMP2++ mice (Figure 4) These results indicatethat phosphorylation of CRMP2 by Cdk5 is required for thedevelopment of proper dendritic spine density in the adultmouse brain

4 Discussion

Recent studies have demonstrated that Cdk5 substrates areinvolved in the regulation of spine formation Synapticproteins phosphorylated by Cdk5 including ephexin1 [18]WAVE1 [19] CRMP1 [13] TrkB [20] PSD95 [21] drebrin[22] and p70 ribosomal S6 kinase (S6K) [23] have beenshown to play a role in spine formation [13 19 21 23]and maintenance [18 20] However their functions differsuch that some of them are crucial for spine formation [13]and some for spine retraction [18] We recently reportedreductions of dendritic spine densities in hippocampal CA1pyramidal neurons of inducible-p35 cKO p39 KO mice andCA1-p35 cKO p39 KO mice with a p35 deletion in the CA1region of the hippocampus after P17 [12] We also reportedreduction of spine densities in cerebral layer V neurons andhippocampal CA1 pyramidal neurons in inducible-p35 cKO

4 Neural Plasticity

CRMP2++

CRMP2KIKI

(a)

lowastlowast

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 2 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in CRMP2KIKI mice at P18 (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at P18 Scale bar 10 120583m (b) Reduced dendritic spine densitywas observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in each area fromthree mice in each genotype were analyzed lowast119875 lt 005

GFPpCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 3 Localization of phospho-CRMP2 at dendritic spines of hippocampal CA1 pyramidal neurons (a) Representative images ofimmunostaining of apical dendrites and their branches with phospho-CRMP2(S522) (pCRMP2S522) antibody in hippocampal CA1pyramidal neurons from GFP-M mice Magnified images of the areas indicated in (a) are shown in (a1015840) Scale bar 10 120583m (b) Representativeimages of immunostaining of apical dendrites and their branches with pCRMP2S522 antibody of hippocampal CA1 pyramidal neurons inGFP-M CRMP2KIKI mice Magnified images of the areas indicated in (b) are shown in (b1015840) Scale bar 10 120583m

Neural Plasticity 5

CRMP2++

CRMP2KIKI

(a)

lowastlowast

45

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 4 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in 5 week-old CRMP2KIKI mice (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at 5 weeks old Scale bar 10 120583m (b) Reduced dendritic spinedensity was observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in eacharea from three mice in each genotype were analyzed lowastlowast119875 lt 001

p39 KO mice when we deleted the p35 gene in 4-month-oldanimals [12]These findings indicate that spine formation andmaintenance are dependent on Cdk5 kinase activity in themouse brain

Our previous study showed that Sema3A-induced spinedevelopment is mediated by phosphorylation of CRMP1 byCdk5 [13] Because CRMP1 and CRMP2 have functionalsimilarities in brain development [14] we examined whetherphosphorylation of CRMP2 by Cdk5 is also important forthe development andmaintenance of dendritic spines in vivoCdk5 specifically phosphorylates Ser residue of CRMP2 at522 [10] We previously generated CRMP2KIKI mice to studythe function of Cdk5-mediated CRMP2 phosphorylationby replacing Ser at 522 to Ala [14] Our present analysisof dendritic spine densities in hippocampal CA1 pyramidalneurons in CRMP2KIKI mice at P18 showed reduced spinedensities in these neurons compared to those of controls(Figure 2) Along with our previous study on CRMP1KOmice [13] these results indicate that CRMPs are importantsubstrates of Cdk5 for spine formation The results obtainedin the present study will provide a new insight into theregulatory mechanisms underlying the effect of Cdk5 ondendritic spine density

Our analysis of 5-week-old CRMP2KIKI mice showedfurther reduction of spine densities in hippocampal CA1pyramidal neurons (Figure 4) These results exclude the pos-sibility that reduced spine densities of hippocampal neuronsin CRMP2KIKI mice at P18 (Figure 2) are due to the delayof brain development In the cerebral cortex of macaquemonkeys and humans the number of dendritic spines rapidly

increases after birth and peaks in an early phase of theinfantile period [24] Spine density then decreases duringthe later infantile period and adolescence period to reachthe adult level [25] Decrease of dendritic spine densityduring the transition from puberty to adulthood has alsobeen reported in the mouse hippocampus [26]These studiesindicate ontogenetic similarity between rodent primate andhuman in spine formation and pruning This overshoot-typetime course of spine formation and pruning is attractivefor researchers because it is possibly involved in develop-mental and psychiatric disorders [27] Further studies arealso required for the analysis of the involvement of CRMP2phosphorylation in spine pruning and maintenance

Cdk5 and its activator p35 play multiple roles in braindevelopment especially in neuronal migration [28] Emerg-ing evidence suggests that Cdk5p35 is also involved insynaptic plasticity [29] Cdk5p35 is localized at neuronalsynapses and phosphorylates many synaptic proteins [21 30ndash33] Furthermore the induction of synaptic plasticity andspatial learning are impaired in Cdk5p35 mutant mice [34ndash36] The role of Cdk5 in synaptic plasticity and learning wasinitially studied using Cdk5 inhibitors which showed inhibi-tion of hippocampal LTP induction and context-dependentfear conditioning [30 37] We have previously reported theimpairment of long-term depression (LTD) induction andspatial learning and memory in p35 KO mice [36] Ourrecent study of p35 conditional KO (cKO) mice which lackhistological abnormalities in the brain also showed impair-ment of spatial learning and memory and LTD induction[38] Importantly electrophysiological analysis of hippocam-pal slices from p35 cKO mice revealed reduced synaptic

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 3

SynaptophysinpCRMP2S522

PSD95pCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 1 Subcellular localization of phospho-CRMP2 in cultured hippocampal neurons (a) Immunocytochemistry with anti-phospho-CRMP2 and synaptophysin antibodies Higher magnification is shown in (a1015840) (b) Immunocytochemistry with anti-phospho-CRMP2 andPSD95 antibodies in cultured rat hippocampal neurons 28 days in vitro (DIV) Merged images are shown Higher magnification shown in(b1015840) Scale bar 20 120583m

contrast its immunoreactivity was very low in those of GFP-M CRMP2KIKI double mutant which is attributable to across reactivity of this antibody to pCRMP1 [14]These resultssuggest that Cdk5 phosphorylates CRMP2 in dendritic spinesof hippocampal CA1 pyramidal neurons in the mouse brain

34 Reduced Spine Densities of Hippocampal CA1 PyramidalNeurons in 5-Week-Old CRMP2119870119868119870119868 Mice We examineddendritic spine density by Golgi staining in 5-week-oldCRMP2KIKI mice Golgi staining of forebrain slices showeda reduction in the numbers of spines in hippocampal CA1pyramidal neurons in CRMP2KIKI mice compared withthose in CRMP2++ mice (Figure 4) These results indicatethat phosphorylation of CRMP2 by Cdk5 is required for thedevelopment of proper dendritic spine density in the adultmouse brain

4 Discussion

Recent studies have demonstrated that Cdk5 substrates areinvolved in the regulation of spine formation Synapticproteins phosphorylated by Cdk5 including ephexin1 [18]WAVE1 [19] CRMP1 [13] TrkB [20] PSD95 [21] drebrin[22] and p70 ribosomal S6 kinase (S6K) [23] have beenshown to play a role in spine formation [13 19 21 23]and maintenance [18 20] However their functions differsuch that some of them are crucial for spine formation [13]and some for spine retraction [18] We recently reportedreductions of dendritic spine densities in hippocampal CA1pyramidal neurons of inducible-p35 cKO p39 KO mice andCA1-p35 cKO p39 KO mice with a p35 deletion in the CA1region of the hippocampus after P17 [12] We also reportedreduction of spine densities in cerebral layer V neurons andhippocampal CA1 pyramidal neurons in inducible-p35 cKO

4 Neural Plasticity

CRMP2++

CRMP2KIKI

(a)

lowastlowast

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 2 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in CRMP2KIKI mice at P18 (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at P18 Scale bar 10 120583m (b) Reduced dendritic spine densitywas observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in each area fromthree mice in each genotype were analyzed lowast119875 lt 005

GFPpCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 3 Localization of phospho-CRMP2 at dendritic spines of hippocampal CA1 pyramidal neurons (a) Representative images ofimmunostaining of apical dendrites and their branches with phospho-CRMP2(S522) (pCRMP2S522) antibody in hippocampal CA1pyramidal neurons from GFP-M mice Magnified images of the areas indicated in (a) are shown in (a1015840) Scale bar 10 120583m (b) Representativeimages of immunostaining of apical dendrites and their branches with pCRMP2S522 antibody of hippocampal CA1 pyramidal neurons inGFP-M CRMP2KIKI mice Magnified images of the areas indicated in (b) are shown in (b1015840) Scale bar 10 120583m

Neural Plasticity 5

CRMP2++

CRMP2KIKI

(a)

lowastlowast

45

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 4 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in 5 week-old CRMP2KIKI mice (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at 5 weeks old Scale bar 10 120583m (b) Reduced dendritic spinedensity was observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in eacharea from three mice in each genotype were analyzed lowastlowast119875 lt 001

p39 KO mice when we deleted the p35 gene in 4-month-oldanimals [12]These findings indicate that spine formation andmaintenance are dependent on Cdk5 kinase activity in themouse brain

Our previous study showed that Sema3A-induced spinedevelopment is mediated by phosphorylation of CRMP1 byCdk5 [13] Because CRMP1 and CRMP2 have functionalsimilarities in brain development [14] we examined whetherphosphorylation of CRMP2 by Cdk5 is also important forthe development andmaintenance of dendritic spines in vivoCdk5 specifically phosphorylates Ser residue of CRMP2 at522 [10] We previously generated CRMP2KIKI mice to studythe function of Cdk5-mediated CRMP2 phosphorylationby replacing Ser at 522 to Ala [14] Our present analysisof dendritic spine densities in hippocampal CA1 pyramidalneurons in CRMP2KIKI mice at P18 showed reduced spinedensities in these neurons compared to those of controls(Figure 2) Along with our previous study on CRMP1KOmice [13] these results indicate that CRMPs are importantsubstrates of Cdk5 for spine formation The results obtainedin the present study will provide a new insight into theregulatory mechanisms underlying the effect of Cdk5 ondendritic spine density

Our analysis of 5-week-old CRMP2KIKI mice showedfurther reduction of spine densities in hippocampal CA1pyramidal neurons (Figure 4) These results exclude the pos-sibility that reduced spine densities of hippocampal neuronsin CRMP2KIKI mice at P18 (Figure 2) are due to the delayof brain development In the cerebral cortex of macaquemonkeys and humans the number of dendritic spines rapidly

increases after birth and peaks in an early phase of theinfantile period [24] Spine density then decreases duringthe later infantile period and adolescence period to reachthe adult level [25] Decrease of dendritic spine densityduring the transition from puberty to adulthood has alsobeen reported in the mouse hippocampus [26]These studiesindicate ontogenetic similarity between rodent primate andhuman in spine formation and pruning This overshoot-typetime course of spine formation and pruning is attractivefor researchers because it is possibly involved in develop-mental and psychiatric disorders [27] Further studies arealso required for the analysis of the involvement of CRMP2phosphorylation in spine pruning and maintenance

Cdk5 and its activator p35 play multiple roles in braindevelopment especially in neuronal migration [28] Emerg-ing evidence suggests that Cdk5p35 is also involved insynaptic plasticity [29] Cdk5p35 is localized at neuronalsynapses and phosphorylates many synaptic proteins [21 30ndash33] Furthermore the induction of synaptic plasticity andspatial learning are impaired in Cdk5p35 mutant mice [34ndash36] The role of Cdk5 in synaptic plasticity and learning wasinitially studied using Cdk5 inhibitors which showed inhibi-tion of hippocampal LTP induction and context-dependentfear conditioning [30 37] We have previously reported theimpairment of long-term depression (LTD) induction andspatial learning and memory in p35 KO mice [36] Ourrecent study of p35 conditional KO (cKO) mice which lackhistological abnormalities in the brain also showed impair-ment of spatial learning and memory and LTD induction[38] Importantly electrophysiological analysis of hippocam-pal slices from p35 cKO mice revealed reduced synaptic

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

4 Neural Plasticity

CRMP2++

CRMP2KIKI

(a)

lowastlowast

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 2 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in CRMP2KIKI mice at P18 (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at P18 Scale bar 10 120583m (b) Reduced dendritic spine densitywas observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in each area fromthree mice in each genotype were analyzed lowast119875 lt 005

GFPpCRMP2S522

(a)

(b)

(a998400)

(b998400)

Figure 3 Localization of phospho-CRMP2 at dendritic spines of hippocampal CA1 pyramidal neurons (a) Representative images ofimmunostaining of apical dendrites and their branches with phospho-CRMP2(S522) (pCRMP2S522) antibody in hippocampal CA1pyramidal neurons from GFP-M mice Magnified images of the areas indicated in (a) are shown in (a1015840) Scale bar 10 120583m (b) Representativeimages of immunostaining of apical dendrites and their branches with pCRMP2S522 antibody of hippocampal CA1 pyramidal neurons inGFP-M CRMP2KIKI mice Magnified images of the areas indicated in (b) are shown in (b1015840) Scale bar 10 120583m

Neural Plasticity 5

CRMP2++

CRMP2KIKI

(a)

lowastlowast

45

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 4 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in 5 week-old CRMP2KIKI mice (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at 5 weeks old Scale bar 10 120583m (b) Reduced dendritic spinedensity was observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in eacharea from three mice in each genotype were analyzed lowastlowast119875 lt 001

p39 KO mice when we deleted the p35 gene in 4-month-oldanimals [12]These findings indicate that spine formation andmaintenance are dependent on Cdk5 kinase activity in themouse brain

Our previous study showed that Sema3A-induced spinedevelopment is mediated by phosphorylation of CRMP1 byCdk5 [13] Because CRMP1 and CRMP2 have functionalsimilarities in brain development [14] we examined whetherphosphorylation of CRMP2 by Cdk5 is also important forthe development andmaintenance of dendritic spines in vivoCdk5 specifically phosphorylates Ser residue of CRMP2 at522 [10] We previously generated CRMP2KIKI mice to studythe function of Cdk5-mediated CRMP2 phosphorylationby replacing Ser at 522 to Ala [14] Our present analysisof dendritic spine densities in hippocampal CA1 pyramidalneurons in CRMP2KIKI mice at P18 showed reduced spinedensities in these neurons compared to those of controls(Figure 2) Along with our previous study on CRMP1KOmice [13] these results indicate that CRMPs are importantsubstrates of Cdk5 for spine formation The results obtainedin the present study will provide a new insight into theregulatory mechanisms underlying the effect of Cdk5 ondendritic spine density

Our analysis of 5-week-old CRMP2KIKI mice showedfurther reduction of spine densities in hippocampal CA1pyramidal neurons (Figure 4) These results exclude the pos-sibility that reduced spine densities of hippocampal neuronsin CRMP2KIKI mice at P18 (Figure 2) are due to the delayof brain development In the cerebral cortex of macaquemonkeys and humans the number of dendritic spines rapidly

increases after birth and peaks in an early phase of theinfantile period [24] Spine density then decreases duringthe later infantile period and adolescence period to reachthe adult level [25] Decrease of dendritic spine densityduring the transition from puberty to adulthood has alsobeen reported in the mouse hippocampus [26]These studiesindicate ontogenetic similarity between rodent primate andhuman in spine formation and pruning This overshoot-typetime course of spine formation and pruning is attractivefor researchers because it is possibly involved in develop-mental and psychiatric disorders [27] Further studies arealso required for the analysis of the involvement of CRMP2phosphorylation in spine pruning and maintenance

Cdk5 and its activator p35 play multiple roles in braindevelopment especially in neuronal migration [28] Emerg-ing evidence suggests that Cdk5p35 is also involved insynaptic plasticity [29] Cdk5p35 is localized at neuronalsynapses and phosphorylates many synaptic proteins [21 30ndash33] Furthermore the induction of synaptic plasticity andspatial learning are impaired in Cdk5p35 mutant mice [34ndash36] The role of Cdk5 in synaptic plasticity and learning wasinitially studied using Cdk5 inhibitors which showed inhibi-tion of hippocampal LTP induction and context-dependentfear conditioning [30 37] We have previously reported theimpairment of long-term depression (LTD) induction andspatial learning and memory in p35 KO mice [36] Ourrecent study of p35 conditional KO (cKO) mice which lackhistological abnormalities in the brain also showed impair-ment of spatial learning and memory and LTD induction[38] Importantly electrophysiological analysis of hippocam-pal slices from p35 cKO mice revealed reduced synaptic

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 5

CRMP2++

CRMP2KIKI

(a)

lowastlowast

45

40

35

30

25

20

15

10

5

0

Num

ber o

f spi

nes

CRMP2++ CRMP2KIKI

(50120583

m)

(b)

Figure 4 Reduction of dendritic spine density in hippocampal CA1 pyramidal neurons in 5 week-old CRMP2KIKI mice (a) Representativephotographs of dendritic segments of hippocampal CA1 pyramidal neurons at 5 weeks old Scale bar 10 120583m (b) Reduced dendritic spinedensity was observed in hippocampal CA1 pyramidal neurons of CRMP2KIKI mice compared to those of control mice 50 neurons in eacharea from three mice in each genotype were analyzed lowastlowast119875 lt 001

p39 KO mice when we deleted the p35 gene in 4-month-oldanimals [12]These findings indicate that spine formation andmaintenance are dependent on Cdk5 kinase activity in themouse brain

Our previous study showed that Sema3A-induced spinedevelopment is mediated by phosphorylation of CRMP1 byCdk5 [13] Because CRMP1 and CRMP2 have functionalsimilarities in brain development [14] we examined whetherphosphorylation of CRMP2 by Cdk5 is also important forthe development andmaintenance of dendritic spines in vivoCdk5 specifically phosphorylates Ser residue of CRMP2 at522 [10] We previously generated CRMP2KIKI mice to studythe function of Cdk5-mediated CRMP2 phosphorylationby replacing Ser at 522 to Ala [14] Our present analysisof dendritic spine densities in hippocampal CA1 pyramidalneurons in CRMP2KIKI mice at P18 showed reduced spinedensities in these neurons compared to those of controls(Figure 2) Along with our previous study on CRMP1KOmice [13] these results indicate that CRMPs are importantsubstrates of Cdk5 for spine formation The results obtainedin the present study will provide a new insight into theregulatory mechanisms underlying the effect of Cdk5 ondendritic spine density

Our analysis of 5-week-old CRMP2KIKI mice showedfurther reduction of spine densities in hippocampal CA1pyramidal neurons (Figure 4) These results exclude the pos-sibility that reduced spine densities of hippocampal neuronsin CRMP2KIKI mice at P18 (Figure 2) are due to the delayof brain development In the cerebral cortex of macaquemonkeys and humans the number of dendritic spines rapidly

increases after birth and peaks in an early phase of theinfantile period [24] Spine density then decreases duringthe later infantile period and adolescence period to reachthe adult level [25] Decrease of dendritic spine densityduring the transition from puberty to adulthood has alsobeen reported in the mouse hippocampus [26]These studiesindicate ontogenetic similarity between rodent primate andhuman in spine formation and pruning This overshoot-typetime course of spine formation and pruning is attractivefor researchers because it is possibly involved in develop-mental and psychiatric disorders [27] Further studies arealso required for the analysis of the involvement of CRMP2phosphorylation in spine pruning and maintenance

Cdk5 and its activator p35 play multiple roles in braindevelopment especially in neuronal migration [28] Emerg-ing evidence suggests that Cdk5p35 is also involved insynaptic plasticity [29] Cdk5p35 is localized at neuronalsynapses and phosphorylates many synaptic proteins [21 30ndash33] Furthermore the induction of synaptic plasticity andspatial learning are impaired in Cdk5p35 mutant mice [34ndash36] The role of Cdk5 in synaptic plasticity and learning wasinitially studied using Cdk5 inhibitors which showed inhibi-tion of hippocampal LTP induction and context-dependentfear conditioning [30 37] We have previously reported theimpairment of long-term depression (LTD) induction andspatial learning and memory in p35 KO mice [36] Ourrecent study of p35 conditional KO (cKO) mice which lackhistological abnormalities in the brain also showed impair-ment of spatial learning and memory and LTD induction[38] Importantly electrophysiological analysis of hippocam-pal slices from p35 cKO mice revealed reduced synaptic

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

6 Neural Plasticity

transmission in hippocampal CA1 pyramidal neurons [38]Since we observed reduced spine densities of hippocampalCA1 pyramidal neurons inCRMP2KIKImice further electro-physiological studies of hippocampal synaptic plasticity andbehavioral analysis in CRMP2KIKI mice will provide furtherknowledge of the significance of Cdk5-mediated CRMP2phosphorylation in synaptic plasticity and in learning andmemory

5 Conclusions

CRMP2 is phosphorylated in dendritic spines of rodent hip-pocampal neurons in vitro and in vivo When we eliminatedCdk5-mediated phosphorylation of CRMP2 at S522 in themouse brain the densities of dendritic spines of hippocampalneurons were reduced in the mouse brain These results sug-gest the regulation of spine density of hippocampal neuronsby CRMP2 phosphorylation

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Jin and Kodai Sasamoto contributed equally to thiswork

Acknowledgments

The authors thank Dr Joshua R Sanes for GFP-M transgenicmice This work was supported by grants from Grant-in-Aidfor JSPS KAKENHI Grant no 26430043 (Toshio Ohshima)

References

[1] Y Goshima F Nakamura P Strittmatter and S M StrittmatterldquoCollapsin-induced growth cone collapse mediated by an intra-cellular protein related to UNC-33rdquo Nature vol 376 no 6540pp 509ndash514 1995

[2] L-HWang and S M Strittmatter ldquoA family of rat CRMP genesis differentially expressed in the nervous systemrdquoThe Journal ofNeuroscience vol 16 no 19 pp 6197ndash6207 1996

[3] M Fukada I Watakabe J Yuasa-Kawada et al ldquoMolecularcharacterization of CRMP5 a novel member of the collapsinresponse mediator protein familyrdquo The Journal of BiologicalChemistry vol 275 no 48 pp 37957ndash37965 2000

[4] R Inatome T Tsujimura T Hitomi et al ldquoIdentification ofCRAM a novel unc-33 gene family protein that associates withCRMP3 and protein-tyrosine kinase(s) in the developing ratbrainrdquo The Journal of Biological Chemistry vol 275 no 35 pp27291ndash27302 2000

[5] J Yuasa-Kawada R Suzuki F Kano T Ohkawara M Murataand M Noda ldquoAxonal morphogenesis controlled by antagonis-tic roles of two CRMP subtypes in microtubule organizationrdquoEuropean Journal of Neuroscience vol 17 no 11 pp 2329ndash23432003

[6] Y Fukata T J Itoh T Kimura et al ldquoCRMP-2 binds to tubulinheterodimers to promote microtubule assemblyrdquo Nature CellBiology vol 4 no 8 pp 583ndash591 2002

[7] N Arimura C Menager Y Kawano et al ldquoPhosphorylationby Rho kinase regulates CRMP-2 activity in growth conesrdquoMolecular and Cellular Biology vol 25 no 22 pp 9973ndash99842005

[8] M Varrin-Doyer A Nicolle R Marignier et al ldquoHuman Tlymphotropic virus type 1 increases T lymphocyte migration byrecruiting the cytoskeleton organizer CRMP2rdquo Journal ofImmunology vol 188 no 3 pp 1222ndash1233 2012

[9] M Tan C Cha Y Ye et al ldquoCRMP4 and CRMP2 interactto coordinate cytoskeleton dynamics regulating growth conedevelopment and axon elongationrdquo Neural Plasticity vol 2015Article ID 947423 13 pages 2015

[10] Y Uchida T Ohshima Y Sasaki et al ldquoSemaphorin3A sig-nalling is mediated via sequential Cdk5 and GSK3beta phos-phorylation of CRMP2 implication of common phosphory-lating mechanism underlying axon guidance and Alzheimerrsquosdiseaserdquo Genes to Cells vol 10 no 2 pp 165ndash179 2005

[11] T Yoshimura Y Kawano N Arimura S Kawabata A KikuchiandK Kaibuchi ldquoGSK-3120573 regulates phosphorylation of CRMP-2 and neuronal polarityrdquo Cell vol 120 no 1 pp 137ndash149 2005

[12] N Mita X He K Sasamoto T Mishiba and T OhshimaldquoCyclin-dependen kinase 5 regulates dendritic spine formationand maintenance of cortical neuron in the mouse brainrdquoCerebral Cortex 2014

[13] N Yamashita A Morita Y Uchida et al ldquoRegulation of spinedevelopment by semaphorin3A through cyclin-dependentkinase 5 phosphorylation of collapsin response mediator pro-tein 1rdquo The Journal of Neuroscience vol 27 no 46 pp 12546ndash12554 2007

[14] N Yamashita TOhshima FNakamura et al ldquoPhosphorylationof CRMP2 (collapsin response mediator protein 2) is involvedin proper dendritic field organizationrdquo The Journal of Neuro-science vol 32 no 4 pp 1360ndash1365 2012

[15] G Feng R H Mellor M Bernstein et al ldquoImaging neuronalsubsets in transgenic mice expressing multiple spectral variantsof GFPrdquo Neuron vol 28 no 1 pp 41ndash51 2000

[16] K Goslin H Asmussen andG Banker ldquoRat hippocampal neu-rons in low-density culturerdquo inCulturing Nerve Cells G Bankerand K Goslin Eds pp 339ndash370 MIT Press Cambridge UK1998

[17] E Niisato J Nagai N Yamashita et al ldquoCRMP4 suppressesapical dendrite bifurcation of CA1 pyramidal neurons in themouse hippocampusrdquo Developmental Neurobiology vol 72 no11 pp 1447ndash1457 2012

[18] W-Y Fu Y Chen M Sahin et al ldquoCdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanismrdquo Nature Neuroscience vol 10 no 1 pp67ndash76 2007

[19] Y Kim J Y Sung I Ceglia et al ldquoPhosphorylation of WAVE1regulates actin polymerization and dendritic spine morphol-ogyrdquo Nature vol 442 no 7104 pp 814ndash817 2006

[20] K-O Lai A S L Wong M-C Cheung et al ldquoTrkB phospho-rylation by Cdk5 is required for activity-dependent structuralplasticity and spatial memoryrdquo Nature Neuroscience vol 15 no11 pp 1506ndash1515 2012

[21] M A Morabito M Sheng and L-H Tsai ldquoCyclin-dependentkinase 5 phosphorylates the N-terminal domain of the postsy-naptic density protein PSD-95 in neuronsrdquo Journal of Neuro-science vol 24 no 4 pp 865ndash876 2004

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 7

[22] K Tanabe H Yamazaki Y Inaguma et al ldquoPhosphorylation ofdrebrin by cyclin-dependent kinase 5 and its role in neuronalmigrationrdquo PLoS ONE vol 9 no 3 Article ID e92291 2014

[23] K O Lai Z Liang E Fei H Huang and N Y Ip ldquoCdk5-dependent phosphorylation of p70 ribosomal S6 kinase (S6K) isrequired for dendritic spine morphogenesisrdquo The Journal ofBiological Chemistry vol 290 no 23 pp 14637ndash14646 2015

[24] P R Huttenlocher ldquoMorphometric study of human cerebralcortex developmentrdquo Neuropsychologia vol 28 no 6 pp 517ndash527 1990

[25] M Missler A Wolff H-J Merker and J R Wolff ldquoPre- andpostnatal development of the primary visual cortex of the com-mon marmoset II Formation remodelling and eliminationof synapses as overlapping processesrdquo Journal of ComparativeNeurology vol 333 no 1 pp 53ndash67 1993

[26] G Meyer R Ferres-Torres and M Mas ldquoThe effects of pubertyand castration on hippocampal dendritic spines of mice AGolgi studyrdquo Brain Research vol 155 no 1 pp 108ndash112 1978

[27] P Penzes M E Cahill K A Jones J-E Vanleeuwen and KM Woolfrey ldquoDendritic spine pathology in neuropsychiatricdisordersrdquoNature Neuroscience vol 14 no 3 pp 285ndash293 2011

[28] T Ohshima J M Ward C-G Huh et al ldquoTargeted disruptionof the cyclin-dependent kinase 5 gene results in abnormalcorticogenesis neuronal pathology and perinatal deathrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 93 no 20 pp 11173ndash11178 1996

[29] K-O Lai and N Y Ip ldquoRecent advances in understanding theroles of Cdk5 in synaptic plasticityrdquo Biochimica et BiophysicaActa vol 1792 no 8 pp 741ndash745 2009

[30] B-S Li M-K Sun L Zhang et al ldquoRegulation of NMDAreceptors by cyclin-dependent kinase-5rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 22 pp 12742ndash12747 2001

[31] M Matsubara M Kusubata K Ishiguro T Uchida K Titaniand H Taniguchi ldquoSite-specific phosphorylation of synapsin Iby mitogen-activated protein kinase and Cdk5 and its effectson physiological functionsrdquo Journal of Biological Chemistry vol271 no 35 pp 21108ndash21113 1996

[32] S C Su J Seo J Q Pan et al ldquoRegulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5rdquo Neuron vol 75 no 4 pp 675ndash687 2012

[33] K Tomizawa S Sunada Y-F Lu et al ldquoCophosphorylationof amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesiclesrdquo Journal of CellBiology vol 163 no 4 pp 813ndash824 2003

[34] J-S Guan S C Su J Gao et al ldquoCdk5 is required for memoryfunction and hippocampal plasticity via the cAMP signalingpathwayrdquo PLoS ONE vol 6 no 9 Article ID e25735 2011

[35] A H Hawasli D R Benavides C Nguyen et al ldquoCyclin-dependent kinase 5 governs learning and synaptic plasticity viacontrol of NMDAR degradationrdquo Nature Neuroscience vol 10no 7 pp 880ndash886 2007

[36] T Ohshima H Ogura K Tomizawa et al ldquoImpairment of hip-pocampal long-term depression and defective spatial learningandmemory in p35minusminusmicerdquo Journal of Neurochemistry vol 94no 4 pp 917ndash925 2005

[37] A Fischer F Sananbenesi C Schrick J Spiess and J RadulovicldquoCyclin-dependent kinase 5 is required for associative learningrdquoJournal of Neuroscience vol 22 no 9 pp 3700ndash3707 2002

[38] T Mishiba M Tanaka N Mita et al ldquoCdk5p35 functions asa crucial regulator of spatial learning and memoryrdquo MolecularBrain vol 7 article 82 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014