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Neuroscience 161 (2009) 978–987

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YCLIN-DEPENDENT KINASE 5 ACTIVATOR p35 OVER-EXPRESSIONND AMYLOID BETA SYNERGISM INCREASE APOPTOSIS IN

ULTURED NEURONAL CELLS

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. UTRERAS,a R. MACCIONIb AND

. GONZÁLEZ-BILLAULTa*

Laboratory of Cell and Neuronal Dynamics, Department of Biologynd Institute for Cell Dynamics and Biotechnology, Faculty of Sci-nces, Universidad de Chile, Las Palmeras 3425, 780-0024 Santiago,hile

Laboratory of Cellular and Molecular Neurosciences, Faculty of Sci-nces and Departments of Neurological Sciences, Faculty of Medi-ine, Universidad de Chile, Santiago, Chile

bstract—Alzheimer disease (AD) is a neurodegenerativeisorder characterized by neuronal loss, dementia and pain.wo main protein aggregates, extracellular (senile plaques,P) and intracellular (neurofibrillary tangles, NFT), are asso-iated with AD. NFT are mainly composed of hyperphospho-ylated microtubule-associated protein tau. Nowadays sev-ral protein kinases have been implicated in the phosphory-

ation of tau, including glycogen synthase kinase 3 betaGSK3�), MAP kinase, protein kinase A and cyclin-dependentinase 5 (Cdk5). A deregulation in the activity of Cdk5 haseen postulated to participate in the abnormal tau hyper-hosphorylation in AD. Activation of Cdk5 occurs after itsssociation with p35, a neuron-specific activator, predomi-antly in the nervous system. Therefore, in this study wesed the tetracycline transactivator system to increase p35/FP in neuronal cells, treated with amyloid beta 1-42 (A�1-42)eptide. These cells showed an increase of terminal deoxy-ucleotidyl transferase dUTP nick end labeling (TUNEL) andleaved caspase-3 staining, indicating increased apoptosisf neuronal cells. This effect could be reversed by the addi-ion of tetracycline in the culture medium, suggesting syner-istic effects of p35 over-expression and A� treatment in thepoptosis of neuronal cells. These results represent a linkageetween amyloidogenic and cdk5 pathways leading to apo-tosis of neuronal cells. © 2009 IBRO. Published by Elseviertd. All rights reserved.

ey words: cleaved caspase-3, Alzheimer’s disease, apopto-is, Tet-off, hippocampal neurons, neuro-2a cell line.

lzheimer disease (AD) corresponds with one of the mostommon types of senile dementia in older adults. AD isharacterized by cognitive disorders associated with a lossf memory and orientation, and deterioration of the intel-

ectual capacity of patients (Selkoe, 1989). Two major

Corresponding author. Tel: �56-2-9787-442; fax: �56-2-2712-983.-mail address: chrgonza@uchile.cl (C. Gonzalez-Billault).bbreviations: AD, Alzheimer disease; A�, �-amyloid peptide; Cdk5,yclin-dependent kinase 5; CFP, cyan fluorescent protein; CIP, cyclin-ependent kinase 5 inhibitor peptide; CMV, cytomegalovirus; EGFP,nhanced green fluorescent protein; NFT, neurofibrillary tangles;BST, 5% nonfat dry milk in PBS with 0.05% Tween-20; tTA, tetracy-

cline dependent transactivator; TUNEL, terminal deoxynucleotidylransferase dUTP nick end labeling.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.04.002

978

istological lesions are observed in AD brains: at the ex-racellular level, senile plaques (SP) are formed by thebrillary �-amyloid peptide (A�) and at the intracellular

evel neurofibrillary tangles (NFTs) composed of pairedelical filaments (PHFs) of the hyperphosphorylated micro-ubule-associated protein tau are formed (Selkoe, 1989).everal protein kinases have been implicated in the phos-horylation of tau protein to date, including glycogen syn-hase kinase 3 beta (GSK3�) (Hong et al., 1997), MAPinase (Mandelkow et al., 1993), protein kinase A (Schnei-er et al., 1999) and, more recently, cyclin-dependentinase 5 (Cdk5) (Alvarez et al., 1999, 2001; Patrick et al.,999).

Cdk5 is a member of the cyclin-dependent kinase fam-ly of serine/threonine kinases, most of which are keyegulators of the cell cycle (Dhavan and Tsai, 2001). Cdk5s mainly active in post-mitotic neurons (Lew et al., 1994;sai et al., 1994), where its activators p35 and p39 arepecifically expressed (Lew et al., 1994; Tsai et al., 1994;hariwala and Rajadhyaksha, 2008). Among its sub-trates, cytoskeletal elements, signaling molecules andegulatory proteins are found (Dhavan and Tsai, 2001;hariwala and Rajadhyaksha, 2008), Pareek and Kulkarni

2006). Deregulation of Cdk5 has been implicated in hu-an neurodegenerative disorders and murine models of

uman neurodegeneration (Dhavan and Tsai, 2001; Mac-ioni et al., 2001; Cruz and Tsai, 2004). Under some cir-umstances p35 protein may be proteolytically processedo give rise to a shorter protein, p25, due to increasedalpain activity (Lee et al., 2000). A potential relationshipetween Cdk5/p25 and the pathology of NFTs has beenroposed based on high levels of p25 detected in therains of patients with AD (Patrick et al., 1999; Tseng et al.,002). A� exposure, or agents that cause an influx ofxtracellular calcium, may induce proteolysis of p35 to p25y activation of calpain (Alvarez et al., 1999; Lee et al.,000). Thus, p25 had been implicated to be responsible forau hyperphosphorylation (Ahlijanian et al., 2000; Noble etl., 2003; Patzke et al., 2003) although some reports havehown this does not necessarily increase tau phosphory-

ation (Kerokoski et al., 2002). Conversely, A� toxicity cane reduced by Cdk5 inhibitors, such as butyrolactone (Al-arez et al., 1999, 2001; Lee et al., 2000; Munoz et al.,000). The linkage between the extracellular A� and the

ncrease of Cdk5 activity was evaluated by conditionallyncreasing the levels of p35 in neuronal cell lines andrimary hippocampal neurons. Our results showed that35 over-expression in neuroblastoma neuro-2a cells in-

reases Cdk5 activity, as well as increasing apoptosis in

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E. Utreras et al. / Neuroscience 161 (2009) 978–987 979

ells treated with A�. Consistently, hippocampal neuronsver-expressing p35/GFP display increased terminal deoxy-ucleotidyl transferase dUTP nick end labeling (TUNEL) andleaved caspase-3 positive staining. Caspase-3 staining waseversed to control values after the addition of tetracycline tourn off the conditional p35 over-expression. Altogether theseesults suggest a synergism between Cdk5/p35 complex andhe amyloidogenic pathway.

EXPERIMENTAL PROCEDURES

aterials

istone H1 and �-tubulin antibody were obtained from Sigma (St.ouis, MO, USA). Protein quantification reagents were obtainedrom Bio-Rad Laboratories (Hercules, CA, USA), and SuperSignal

est Pico chemiluminescent substrate for Western blot analysisere purchased from Thermo Scientific (Rockford, IL, USA). A�as obtained from Global Peptide (Fort Collins, CO, USA).

ntibodies

ntibodies to Cdk5 and p35, and secondary antibodies (HRP-onjugated goat anti-mouse, anti-rabbit and anti-goat antibodies)ere obtained from Santa Cruz Biotechnology Inc. (Santa Cruz,A, USA). Antibody to enhanced green fluorescent protein

EGFP) was obtained from Clontech (Mountain View, CA, USA).ntibody to cleaved caspase-3 was obtained from Cell Signalingechnology (Beverly, MA, USA).

ell line culture

euro-2a cell line (derived from mouse neuroblastoma) stablyransfected with the tetracycline dependent transactivator (tTA)as cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,

nvitrogen, Carlsbad, CA, USA) supplemented with 10% fetalovine serum (Hyclone Laboratories, Logan, UT, USA) and 250�l/ml) G418 Sulfate (GIBCO, CA, USA). These cells were kindlyrovided by Dr. Javier Diaz-Nido (Centro de Biologia Molecularevero Ochoa, Madrid, Spain).

rimary mouse hippocampal neurons culture

rimary mouse hippocampal neuronal cultures were establishedrom the hippocampus of 18-day-old mouse FVB fetuses accord-ng with methods described previously (Banker and Cowan,977). Briefly, neurons were seeded onto 0.1% polylysine-coatedoverslips in 35-mm plastic Petri dishes to 2�105 cell/cm2 andultured for 4 days in neurobasal medium plus supplement B27Invitrogen).

myloid-� preparation

ynthetic A�42 peptide solutions (�70% HPLC) were prepared asescribed previously (Evans et al., 1995). Briefly, stock solutionsere prepared by dissolving A� in dimethyl sulfoxide (DMSO) at 1

�g/�l). The solution was incubated for 7 days with constanttirring at room temperature. The fibrils were concentrated byentrifugation and resuspended in sterile PBS. Formation of A�brils was confirmed by electronic microscopy.

reparation of the pBI-p35/EGFP plasmid

e constructed a pBI-p35/EGFP vector by inserting a cDNAouse p35 (924 pb) into the pBI-EGFP vector from Clontech.riefly, the cDNA of p35 was amplified with specific primer: p35-heI sense 5=-GAC GCG TAT GGG CAC GGT GCT GTC C-3=

nd p35-MluI antisense 5=-GCG CTA GCG GCT CAC CGA TCC w

GC C-3=. The PCR product was digested with NheI y MluI andirectionally cloned into pBI-EGFP vector within the same restric-ion sites. Positive clones were analyzed by restriction enzymesnd DNA sequencing.

ransient transfection of cell line

euro-2a cells expressing tTA protein were transiently transfectedith 1 �g of pBI-p35/EGFP vector in Opti-MEM with Lipofectamin000 (Invitrogen). After the transfection, cells were treated withnd without 1 (�g/ml) tetracycline for 48 h. Primary cultures ofippocampal neurons were transiently transfected with pBI-p35/GFP, cytomegalovirus (CMV)-tTA and CMV–cyan fluorescentrotein (CFP) vectors with lipofectamine in Opti-MEM (Invitrogen).fter 24 h of transfection, neurons were fixed and permeabilized

or confocal immunofluorescence analyses.

mmunofluorescence analysis

euro-2a cells and hippocampal neurons were seeded over cov-rslips. Cells were fixed in a mix of 4% paraformaldehyde and 4%ucrose for 20 min at 37 °C. Later, the cells were subsequentlyermeabilized in cold methanol and 0.1% PBS–Triton X-100 for0 min each. The cells were blocked in 5% BSA-PBS for 1 h and

ncubated with primary antibodies for 1 h in 1% BSA-PBS. Sec-ndary antibodies coupled to fluorescein isothiocyanate (FITC),hodamine or Alexa Fluor 546 were incubated for 1 h in 1%SA-PBS and cells were analyzed in Confocal LSM 510 micros-opy (Zeiss).

mmunoblot analysis

roteins were extracted with RIPA buffer (Tris 50 mM, pH 7.5,aCl 150 mM, EDTA 5 mM, NP-40 1%, sodium deoxycholate.5% and SDS 0.1%), supplemented with protease inhibitorsPMSF 100 �g/ml, aprotinin 2 �g/ml, leupeptin 2 �M y pepstatin 1g/ml). Protein concentration of the supernatant was determinedy the Bradford Protein Assay (Bio-Rad). Thirty micrograms ofroteins was separated in 6%–15% SDS-PAGE gels and trans-erred to nitrocellulose membranes (Invitrogen). The membranesere soaked in a blocking buffer (5% nonfat dry milk in PBS with.05% Tween-20 (PBST)) for 1 h at room temperature and then

ncubated overnight at 4 °C with the appropriate primary antibodyiluted in 1% nonfat milk in PBST buffer. The membranes wereashed in PBST and incubated for 1 h at room temperature, with

he secondary antibodies diluted in 1% nonfat milk in PBST buffer.mmunoreactivity was detected using SuperSignal West Picohemiluminescent substrate (Thermo Scientific). Membranesere stripped for 30 min at 50 °C in stripping buffer (2% SDS, 100M �-mercaptoethanol and 50 mM Tris, pH 6.8) and retested with

ubulin antibody to normalize the protein loading. Optical densitiesf the bands were quantified by using an image analysis systemith Scion Image Alpha 4.0.3.2 software (Scion Corporation,rederick, MD, USA).

dk5 kinase activity assay

dk5 kinase activity was measured as described (Alvarez et al.,001). Five hundred micrograms of protein from neuro-2a cellsas dissolved in RIPA buffer and immunoprecipitated with anti-dk5 antibody (5 �g). Immunoprecipitated proteins were washed

n kinase buffer (Hepes 50 mM, MgCl2 10 mM, MnCl2 5 mM, DTT�M), mixed with kinase assay mixture (Hepes 50 mM, MgCl2 10M, MnCl2 5 mM, DTT 1 �M, 5 �Ci [�-32P] ATP, and histone H1

2.5 �g/�l) as a substrate), and the kinase activity was quantifieds described (Alvarez et al., 2001).

poptosis analysis

euro-2a cell and hippocampal primary cultures co-transfected

ith pBI-p35/EGFP, CMV-tTA vectors were treated with A�1-42 10

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E. Utreras et al. / Neuroscience 161 (2009) 978–987980

M for 24 h, and the apoptosis level was measured by TUNELsing the kit in situ Cell Death Detection (Roche, Mannheim,ermany), as well as by immunofluorescence of cleaved

aspase-3. The cells were counted and analyzed in a confocalSM 510 microscopy (Zeiss).

eversion of p35 over expression

rimary cultures of hippocampal neurons co-transfected with pBI-35/EGFP, CMV-tTA and CMV-CFP vectors were treated with�1-42 10 �M for 24 h in presence or absence of 1 (�g/ml)

etracycline, and the level of apoptosis was measured by inmuno-ourescence of cleaved caspase-3. The cells were counted andnalyzed in Confocal LSM 510 microscopy (Zeiss).

ig. 1. Generation of p35 conditional over-expression system. (A) Thf seven copies of the palindromic tet operator sequence flankedi-directional promoter is followed by p35 cDNA sequence in one dire

he CMV promoter. Tetracycline or doxycycline can bind to tTA and inaransiently co-transfected with pBI-p35/EGFP and CMV-tTA vectors in

n the culture medium (Tet-OFF, right panel). Phase-contrast is shown in the upntransfected cells.

tatistical analysis

ll experiments were performed in triplicates. Statistical evaluationas done with Graphic Prism software, version 4.0 (GraphPad, Saniego, CA, USA). Significant differences between experiments weressessed by univariate ANOVA (more than two groups) or unpairedtudent’s t-test (two groups), and � was set at 0.05.

RESULTS

eneration of p35 conditional over-expression system

e used a tetracycline inducible system to generate aell-based assay for inducible p35 over-expression. Reg-

is compound for two vectors: pBI-p35/EGFP construct that consistsMV minimum promoter sequences in divergent orientations. ThisEGFP cDNA sequence in the other; and tTA vector under control of

ith subsequent inactivation of p35 and EGFP. (B) Neuro-2a cells werece (Tet-ON, middle panel) or in the presence of tetracycline (1 �g/ml)

is systemby two Cction andctive it wthe absen

per panels and EGFP fluorescence is shown in the lower panel. UT:

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E. Utreras et al. / Neuroscience 161 (2009) 978–987 981

lation of the system is achieved through the tTA, a chi-eric protein composed of the Tet-repressor DNA-bindingomain and the transactivation domain of viral proteinP16 of herpes simplex virus (Gossen and Bujard, 1992).his protein binds specifically to the tetO operator se-uence and induces transcription from an adjacent CMVinimal promoter. The combination of both tTA and the

etO elements thus allows continual transactivation of aiven transgene. Tetracycline and its analogs can bind toTA. When this happens, tTA is prevented from binding toetO, and transcription is inhibited. We generated a plas-id carrying the bi-directional Tet-responsive promoter

Baron et al., 1995), followed by a p35 cDNA in one direc-ion and, in the other direction, a cDNA encoding EGFPFig. 1A). The resulting plasmid termed pBI-p35/EGFPas assayed in transient transfection experiments per-

ormed in neuro-2a cells stably transfected with CMV-tTAn presence or absence of tetracycline (Fig. 1B). Transientransfection efficiency was 48%�5.7%, as determined byGFP intrinsic fluorescence (data not shown). Using im-unofluorescence, we determined that neuro-2a-tTA cellsxpress low levels of p35 protein (arrowheads), but afterransient transfection with pBI-p35/EGFP vector, the p35nd EGFP protein levels increased significantly in theame cells (Fig. 2A, arrows). The overall morphology ofransfected cells was unchanged, as indicated by �-tubulintaining (Fig. 2A). Western blot analysis determined that35 endogen levels were very low in untransfected cellsUT, lane 1). In neuro-2a-tTA cells transfected with pBI-35/EGFP (Tet-ON), the levels of p35 increased in relationo p35 endogen levels (Fig. 2B, compare lanes 1 and 3).

hen we added tetracycline (Tet-OFF), we observed aecrease in p35 expression, as compared with cells trans-ected without tetracycline (Fig. 2B, compare lanes 3 and). Moreover, EGFP protein levels increased in neuro-2a-TA cells transfected with pBI-p35/EGFP and decreased toasal level in the presence of tetracycline (Fig. 2B, lanes 3nd 4). Because p35 protein is a limiting factor that regu-

ates the Cdk5 kinase activity (Takahashi et al., 2005;treras et al., 2009), we analyzed whether the p35 over-xpression resulted in an increase of Cdk5 activity. We

mmunoprecipitated Cdk5 protein from neuro-2a-tTA cellsransfected with pBI-p35/EGFP, and then assayed kinasectivity by using histone H1 as a substrate (Fig. 3). Cdk5inase activity increased by four in cells transfected withBI-p35/EGFP (Tet-ON), in the absence of tetracycline,nd decreased to basal levels in the presence of tetracy-line (Tet-OFF) (Fig. 3). Together, these results indicatehat the over-expression of p35 causes an increase indk5 kinase activity, and this effect is inhibited by tetracy-line in neuro-2a cells.

35 over-expression synergism with A� treatmentausing apoptosis in neuro-2a cells

euro-2a cells were treated in the presence or absence of� for 24 h and the level of apoptosis was measured byUNEL (Fig. 4A). The A� treatment increased apoptosis

evels compared to UT cells (14.3�1.9 versus 1.0�0.4,

P�0.05). Neuro-2a cells transiently co-transfected with p

BI-p35/EFGP and CMV-tTA, the apoptosis levels did nothange as compared with UT cells (1.6�0.9 versus.0�0.4; P�0,354). In contrast, the percentage of apopto-is was higher in neuro-2a cells Tet-ON treated with A�ompared to cells Tet-ON without A� (21.5�0.9 versus4.3�1.1; P�0.001) (Fig. 4B). Together, these resultsuggest that p35 over-expression promotes death by ap-ptosis in the presence of A� treatment in neuro-2a cells.

35 over-expression synergism with A� treatmentausing apoptosis in primary culture of mouseippocampal neurons

o further test this synergistic effect between p35 over-xpression and A� treatment, we cultured primary hip-ocampal neurons from mice and co-transfected them with

ig. 2. p35 Over-expression in neuro-2a cells. (A) Immunofluores-ence of neuro-2a-tTA cells transiently transfected with pBI-p35/EGFPector. The left panel shows phase-contrast, the middle panel showsGFP fluorescence (green) and the right panel shows p35 or �-tubulin

mmunofluorescence (red). Arrow shows the same cell transientlyransfected with over-expressing p35 and EGFP and arrowheadhows p35 basal expression level in neuro-2a cell. (B) Western blotnalysis shows neuro-2a-tTA transiently transfected with pBI-p35/GFP in the absence (Tet-ON, lane 3) or presence of tetracycline

1 �g/ml) (Tet-OFF, lane 4). Untransfected cells (UT, lane 1) or pBI-GFP-transfected cells (lane 2) were used as control. p35 Was de-

ected with two different antibodies: C19 antibody recognizes the-terminus and N20 antibody recognizes the N-terminus of p35 pro-

ein. Also p25 was recognized with C-19 antibody. Western blottingnalysis against �-tubulin was used as a loading control. For interpre-

ation of the references to color in this figure legend, the reader iseferred to the Web version of this article.

BI-p35/EGFP and CMV-tTA vectors, in the presence and

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E. Utreras et al. / Neuroscience 161 (2009) 978–987982

bsence of A� for 24 h. Immunofluorescence against p35nd EGFP fluorescence showed that both proteins arexpressed in the same neurons and the morphology ofo-transfected neurons seems normal in the absence of� (Fig. 5A). However, in the presence of A�, the mor-hology in some co-transfected neurons was similar toeurons displaying apoptotic signs such as dystrophiceurites (Fig. 5B, arrow). In fact, the axon displayed in-reased varicosities most likely due to cytoskeleton disrup-ion (Fig. 5B). In order to test whether A� treatment in-uces apoptosis in hippocampal neurons, we performedUNEL assay in these cultures (Fig. 6A–E). Primary cul-

ures of hippocampal neurons were very sensitive to apo-tosis, reflected by the fact that basal TUNEL positiveeurons were around 20% (Fig. 6E). In Tet-ON neurons,he percentage of apoptosis increased compared to UTeurons (35.2�3.2 versus 20.0�1.2; P�0.05), but in theresence of tetracycline the percentage of apoptosis de-reased to basal levels in Tet-OFF neurons (20.8�5.1). Inet-ON neurons treated with A�, the levels of apoptosisere increased twofold compared with Tet-ON neuronsithout A� (62.6�7.6 versus 35.2�3.2; P�0.001) (Fig.E). To confirm these results, we used another approach

ig. 3. p35 Over-expression increased Cdk5 activity in neuro-2a cells.A) Neuro-2a-tTA cells were transiently transfected with pBI-p35/GFP for 48 h in the absence (Tet-ON) or presence of tetracycline (1g/ml) (Tet-OFF). Cdk5 was immunoprecipitated with Cdk5 antibody

C-8) and its kinase activity was assayed using histone H1 as aubstrate. Cdk5 Western blot analysis was used as loading control. (B)dk5 activity was four times increased in cells transfected with pBI-35/EGFP compared with untransfected cells (UT) and this increaseas inhibited in the presence of tetracycline. Bars are the mean�SEMf three independent experiments. * P�0.05 by using Student’s test.

o determine apoptosis in hippocampal neurons. We used*t

mmunofluorescence against cleaved caspase-3, a keyrotein involved in execution of apoptosis (Porter and Jänicke,999). A specific antibody against cleaved caspase-3 rec-gnizes a proteolytic fragment 17/19 kDa that is produced

n early stages of apoptosis. Primary cultures of hippocam-al neurons were co-transfected with pBI-p35/EGFP andMV-tTA vectors in the absence (Fig. 7A) or presence of� for 24 h (Fig. 7B). The induction of apoptosis wasvaluated in positive cells for both EGFP and cleavedaspase-3 staining. The co-localization of EGFP andleaved caspase-3 staining was most likely to be found inells treated with A� in compared with cells without A�

63% over 21%, P�0.0033) (Fig. 7C). Together, these

ig. 4. Induction of apoptosis by A� in neuro-2a cells (TUNEL).A) Apoptosis was measured using TUNEL in neuro-2a cells. TUNELeasuring was done in untransfected cells (UT), cells transiently

o-transfected with pBI-p35/EGFP and CMV-tTA vectors in the ab-ence (Tet-ON), or presence of tetracycline (1 �g/ml) (Tet-OFF), or UTells treated with A� (10 �M) (A�), or in Tet-ON cells treated with A�Tet-ON�A�) for 24 hours. (B) The percentage of TUNEL positiveeuro-2a cells does not changed in cells transfected with p35 in thebsence (Tet-ON) or presence of tetracycline (Tet-OFF) comparedith UT cells. In contrast, in neuro-2a cells treated with A� the per-entage of TUNEL positive cells increased significantly compared withT cells (* P�0.001). Also, in Tet-ON cells treated with A� (A��Tet-N) the percentage of TUNEL positive cells increased significantlyompared with Tet-ON cells (** P�0.001). Bars are the mean�SEM ofounting TUNEL positive cells in three independent experiments.

P�0.001 by using ANOVA test with Bonferroni multiple comparison

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E. Utreras et al. / Neuroscience 161 (2009) 978–987 983

esults suggest a synergistic effect between Cdk5/p35omplex and the amyloidogenic pathway.

eversibility of p35 over-expression causes reducedpoptosis in the presence of A� in hippocampaleurons

eversibility of p35 expression was evaluated in hip-ocampal neurons co-transfected with three different vec-ors: pBI-p35/EGFP, CMV-tTA and CMV-CFP. Neuronsriple-transfected in the absence of tetracycline expressesGFP and p35, along with the expression of CFP, indicat-

ng that all the constructs may be expressed at the sameime (Fig. 8A). Hippocampal triple-transfected neuronsere treated with A� in the absence or presence of tetra-ycline. Turning off the p35/GFP expression protects neu-

ig. 5. Morphologic changes in hippocampal neurons transfected withith pBI-p35/EGFP and CMV-tTA in the absence (A) or presence of Auorescence shows that both proteins are expressed in the same neurof A� (A), but in the presence of A� in some EGFP positive neuronsrotein levels decreased in these neurons (B, arrow).

ons against A�-induced apoptosis (compare arrowheads r

ndicating caspase-3 positive and caspase-3 negativeells, Fig. 8B). These results suggest that there is a phys-ological correlation between amyloidogenic pathway andhe increase of Cdk5/p35 activity.

DISCUSSION

n this work we have shown that conditional over-expres-ion of p35 synergized with A� treatment leads to apopto-ic cells’ death. This effect could be reversed by turning offhe over-expression of p35 using tetracycline in our cellulture system. We also showed that p35 over-expression

n neuro-2a cells induced an increase in Cdk5 activity, andhat this effect was inhibited in the presence of tetracycline.urthermore, the treatment with A� in hippocampal neu-

treated with A�. Hippocampal neurons were transiently co-transfected) (B) for 24 h. Immunofluorescence against p35 and EGFP endogene morphology of co-transfected neurons seems normal in the absencehology was similar to neurons in apoptotic processes, and also p35

p35 and� (10 �Mns and th

ons over-expressing p35 produced an increase in the

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E. Utreras et al. / Neuroscience 161 (2009) 978–987984

poptosis determinate by TUNEL and cleaved caspase-3uggesting a synergism between Cdk5/p35 complex andhe amyloidogenic pathway. These results supports theypothesis that A� treatment induces cell death (Alvarez etl., 1999, 2001; Liu et al., 2004; Han et al., 2005). Neuro-oxic effects of A� on primary cultures decrease sharply inhe presence of butyrolactone, a Cdk5 inhibitor. Moreover,he death of neurons was also prevented by using anntisense probe against Cdk5 (Alvarez et al., 1999). Onhe other hand, �-amyloid precursor protein (APP) treat-ent prevents tau hyperphosphorylation via suppressingveractivation of Cdk5 (Han et al., 2005). The increase indk5 activity was associated with tau protein hyperphos-horylation, but this effect was not due to change in the

evels of Cdk5 and p35 mRNA after A� treatment, whichuggests that the increase in Cdk5 activity could be aost-translational mechanism (Alvarez et al., 2001). More-ver, A� toxicity is dependent on the differentiation stages

ig. 6. Induction of apoptosis by A� in hippocampal neurons (TUNELeurons transiently co-transfected with pBI-p35/EGFP and CMV-tTMV-tTA in the presence of tetracycline (1 �g/ml) (Tet-OFF); and (D)

Tet-ON�A�) for 24 h (E) The percentage of TUNEL positive neurons wevels in Tet-OFF cells (* P�0.05). In contrast, in neurons with Tet-Oignificantly compared with neurons with Tet-ON (** P�0.001). Barsndependent experiments. * P�0.05 and ** P�0.001 by using ANOVA

f the neurons. Indeed, only cells containing appreciable c

evels of tau are susceptible to A�-induced toxicity, whichay explain why A� is more toxic to neurons than to other

ell types (Liu et al., 2004).In our study, we show that p35 over-expression in-

uced more Cdk5 activity, which supports the role of thisinase in apoptosis since A� treatment produced an in-rease of cytotoxicity in neuro-2a cells and hippocampaleurons. Although there are differences in susceptibility to� between neuro-2a cells and hippocampal neurons, it islear that the p35 over-expression contributes to increasedeaths due to the effect of the neurotoxic peptide. Theseesults suggest that higher p35 protein levels, and subse-uent activation of Cdk5 activity, are responsible for increas-

ng apoptotic processes. Interestingly, in both neuro-2a cellsnd hippocampal neurons, higher p35 protein levels wereorrelated with an increase in the frequency of apoptoticarkers (TUNEL and cleaved caspase-3). Although the

orrelation is not complete, we can conclude that there is a

sis was measured by TUNEL in (A) neurons untransfected (UT); (B)N); (C) neurons transiently co-transfected with pBI-p35/EGFP andtransiently co-transfected with pBI-p35/EGFP and CMV-tTA plus A�sed in Tet-ON cells in compared with UT cells and decreased to basal

A� (A��Tet-ON) the percentage of TUNEL positive cells increasedmean�SEM of counting TUNEL positive neurons in at least three

h Bonferroni multiple comparison test.

). ApoptoA (Tet-Oneuronsas increaN plus

ausal effect between p35 expression and the susceptibil-

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E. Utreras et al. / Neuroscience 161 (2009) 978–987 985

ty to death which A� treatment induces. The last part ofur experimental design seeks to demonstrate that theeversion of p35 over-expression would have an effect oneath induced by A�. We co-transfected our p35 over-

ig. 7. Induction of apoptosis by A� in hippocampal neurons (cleavedaspase-3). Apoptosis was measured by immunofluorescence againstleaved caspase-3 in hippocampal neurons transiently co-transfectedith pBI-p35/EGFP and CMV-tTA in the absence (A) or presence of� (B) for 24 h. Immunofluorescence against cleaved caspase-3 (red),GFP fluoresce (green), and nucleus (blue), was combined in theerge. (C) Quantification of the frequency of EGFP and cleaved

aspase-3 positive neurons was higher in the presence than in ab-ence of A� (* P�0.05). by using the Fisher test. For interpretation ofhe references to color in this figure legend, the reader is referred tohe Web version of this article.

xpression inducible system and one constitutive CFPht

ector in hippocampal neurons as a transfection control.nterestingly, these experiments showed that neuronshose p35 expression was regulated by tetracycline had arotective effect on neurotoxicity of A�, in detriment to

hose neurons that did not over-express the transgene.he response of neurons to A� does not seem to beomogeneous, suggesting differential accessibility to A� inhe culture. In accordance with these findings, Maloney etl. (2005) showed that the effect of A� (fiber, oligomers oroluble amyloid peptide) over hippocampal neurons was

ig. 8. Reversibility of p35 over-expression cause reduced apoptosisn the presence of A� in hippocampal neurons. (A) Hippocampaleurons were transiently triple-transfected with pBI-p35/EGFP, CMV-

TA and CMV-CFP vectors for 24 h and immunofluorescence against35 (red), EGFP fluorescence (green), and CFP fluorescence (cyan)as done. (B) Hippocampal neurons triple-transfected with pBI-p35/GFP, CMV-tTA and CMV-CFP were treated with A� (10 �g) and

etracycline (1 �g/ml) for 24 h. Immunofluorescence against cleavedaspase-3 showed that most of the cleaved caspase-3 positive neu-ons (arrows) were different from neurons triple-transfected (arrow-

ead). For interpretation of the references to color in this figure legend,

he reader is referred to the Web version of this article.

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E. Utreras et al. / Neuroscience 161 (2009) 978–987986

ot homogeneous. This may also suggest that hippocam-al neurons can display differential response mechanisms

o A� injury.Although the mechanisms involved are not entirely

lear at the present, they may include the participation ofdk5 in the prevention or induction of apoptosis (Eilers etl., 1998; Alvarez et al., 1999, 2001; Patrick et al., 1999; Lit al., 2002, 2003; Weishaupt et al., 2003; Zheng et al.,005; Cheung et al., 2008). Cdk5 can participate in pre-enting apoptosis through phosphorylation of JNK3. In-eed, the activation of JNK3 induces apoptosis (Eilers etl., 1998), but the JNK3 phosphorylation mediated bydk5 negatively regulates the activity of this kinase, pro-

ecting the neurons from death by apoptosis (Li et al.,002). Moreover, Cdk5 regulates Akt activity and cell sur-ival through the neuroregulin-mediated PI3-kinase signal-ng pathway (Li et al., 2003). In addition, Cheung andoworker (2008) found that the reduction or absence ofdk5 activity markedly exacerbated neuronal death

hrough direct phosphorylation of Bcl-2. In contrast, corticaleurons infected with a cyclin-dependent kinase 5 inhibitoreptide (CIP) decreased tau phosphorylation and apopto-is. Indeed, the apoptosis induced by A� in cortical neu-ons was reduced by CIP expression (Zheng et al., 2005).dditionally, other authors relate deregulation of Cdk5 ac-

ivity with an induction of apoptosis (Alvarez et al., 1999,001; Patrick et al., 1999; Weishaupt et al., 2003) Accord-

ng to the hypothesis that Cdk5/p25 complex is a down-tream activator of the cascade of neuronal death in theD, the A� treatment of hippocampal neurons prompted

he disruption of p35 into p25 with a subsequent increasen Cdk5 activity (Lee et al., 2000). Additionally, the inhibi-ion of Cdk5 with butyrolactone protected neurons againsteath (Alvarez et al., 2001). These discrepancies on theotential role of Cdk5 in apoptosis could explain our resultsn the reversion of p35 expression, because the cells thatave not been transfected with transgenes seem to have areater susceptibility to amyloid peptide, compared with

hose transfected and subsequently regulated by tetracy-line. Possibly the early p35 expression, before the rever-ion, could protect neurons from A�, in relation to neuronsot transfected. Finally, the synergism between A� anddk5/p35 complex in the activation of apoptotic pathways

hat trigger neuronal death could be explained by the se-ies of events triggered after A� binding to several recep-ors that leads to increased calcium influx responsible foralpain activation. The position of Cdk5 within the cascadef events remains speculative, but recently new evidenceas arisen which suggests that Cdk5 is located upstreamf the mitochondrial dysfunction. This is because Cdk5 canirectly phosphorylate Bcl-2 and inhibit apoptosis (Cheungt al., 2008), and also because Cdk5 can act as an up-tream regulator of mitochondrial fission, integrating Cdk5nto an established neuronal apoptosis pathway (Meuer etl., 2007).

Our study therefore contributes to the understanding ofhe Cdk5/p35 complex’s participation in the events occur-

ing after A� addition, which lead to apoptosis and possible

ynergism with the amyloidogenic pathway in the inductionf apoptosis in hippocampal neurons.

cknowledgments—We would like to thank Dr. Jose Lucas for theMV-tTA vector and Ms. Lorena Saragoni for their helpful immu-ofluorescence analysis. This work was supported by grants fromondecyt 3010060, 1060040 and Initiative Scientific Millennium

CMP05-001-F to C.G.-B. R.M. was supported by grants fromondecyt and the International Center for Biomedicine.

REFERENCES

hlijanian MK, Barrezueta NX, Williams RD, Jakowski A, Kowsz KP,McCarthy S, Coskran T, Carlo A, Seymour PA, Burkhardt JE,Nelson RB, McNeish JD (2000) Hyperphosphorylated tau andneurofilament and cytoskeletal disruptions in mice overexpressinghuman p25, an activator of cdk5. Proc Natl Acad Sci U S A 97:2910–2915.

lvarez A, Munoz JP, Maccioni RB (2001) A Cdk5-p35 stable complexis involved in the beta-amyloid-induced deregulation of Cdk5 ac-tivity in hippocampal neurons. Exp Cell Res 264:266–274.

lvarez A, Toro R, Caceres A, Maccioni RB (1999) Inhibition of tauphosphorylating protein kinase cdk5 prevents beta-amyloid-in-duced neuronal death. FEBS Lett 459:421–426.

anker GA, Cowan WM (1977) Rat hippocampal neurons in dispersedcell culture. Brain Res 126:397–342.

aron U, Freundlieb S, Gossen M, Bujard H (1995) Co-regulation oftwo gene activities by tetracycline via a bidirectional promoter.Nucleic Acids Res 23:3605–3606.

heung ZH, Gong K, Ip NY (2008) Cyclin-dependent kinase 5 sup-ports neuronal survival through phosphorylation of Bcl-2. J Neuro-sci 28:4872–4877.

ruz JC, Tsai LH (2004) Cdk5 deregulation in the pathogenesis ofAlzheimer’s disease. Trends Mol Med 10:452–458.

hariwala FA, Rajadhyaksha MS (2008) An unusual member of thecdk family: cdk5. Cell Mol Neurobiol 28:351–369.

havan R, Tsai LH (2001) A decade of CDK5. Nat Rev Mol Cell Biol2:749–759.

ilers A, Whitfield J, Babij C, Rubin LL, Ham J (1998) Role of the Junkinase pathway in the regulation of c-Jun expression and apoptosisin sympathetic neurons. J Neurosci 18:1713–1724.

vans KC, Berger EP, Cho CG, Weisgraber KH, Lansbury PT Jr(1995) Apolipoprotein E is a kinetic but not a thermodynamicinhibitor of amyloid formation: implications for the pathogenesisand treatment of Alzheimer disease. Proc Natl Acad Sci U S A92:763–767.

ossen M, Bujard H (1992) Tight control of gene expression in mam-malian cells by tetracycline-responsive promoters. Proc Natl AcadSci U S A 89:5547–5551.

an P, Dou F, Li F, Zhang X, Zhang YW, Zheng H, Lipton SA, Xu H,Liao FF (2005) Suppression of cyclin-dependent kinase 5 activa-tion by amyloid precursor protein: a novel excitoprotective mech-anism involving modulation of tau phosphorylation. J Neurosci25:11542–11552.

ong M, Chen DC, Klein PS, Lee VM (1997) Lithium reduces tauphosphorylation by inhibition of glycogen synthase kinase-3. J BiolChem 272:25326–25332.

erokoski P, Suuronen T, Salminen A, Soininen H, Pirttila T (2002)Cleavage of the cyclin-dependent kinase 5 activator p35 to p25does not induce tau hyperphosphorylation. Biochem Biophys ResCommun 298:693–698.

ee MS, Kwon YT, Li M, Peng J, Friedlander RM, Tsai LH (2000)Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature405:360–364.

ew J, Huang QQ, Qi Z, Winkfein RJ, Aebersold R, Hunt T, Wang JH(1994) A brain-specific activator of cyclin-dependent kinase 5.

Nature 371:423–426.

L

L

L

M

M

M

M

M

N

P

P

P

P

S

S

T

T

T

U

W

Z

E. Utreras et al. / Neuroscience 161 (2009) 978–987 987

i BS, Ma W, Jaffe H, Zheng Y, Takahashi S, Zhang L, Kulkarni AB,Pant HC (2003) Cyclin-dependent kinase-5 is involved in neuregu-lin-dependent activation of phosphatidylinositol 3-kinase and Aktactivity mediating neuronal survival. J Biol Chem 278:35702–35709.

i BS, Zhang L, Takahashi S, Ma W, Jaffe H, Kulkarni AB, Pant HC(2002) Cyclin-dependent kinase 5 prevents neuronal apoptosis bynegative regulation of c-Jun N-terminal kinase 3. EMBO J 21:324–333.

iu T, Perry G, Chan HW, Verdile G, Martins RN, Smith MA, AtwoodCS (2004) Amyloid-beta-induced toxicity of primary neurons isdependent upon differentiation-associated increases in tau andcyclin-dependent kinase 5 expression. J Neurochem 88:554–563.

accioni RB, Otth C, Concha MJP II, Munoz JP (2001) The proteinkinase Cdk5. Structural aspects, roles in neurogenesis and in-volvement in Alzheimer’s pathology. Eur J Biochem 268:1518–1527.

aloney MT, Minamide LS, Kinley AW, Boyle JA, Bamburg JR (2005)Beta-secretase-cleaved amyloid precursor protein accumulates atactin inclusions induced in neurons by stress or amyloid beta: afeedforward mechanism for Alzheimer’s disease. J Neurosci 25:11313–11321.

andelkow EM, Biernat J, Drewes G, Steiner B, Lichtenberg-Kraag B,Wille H, Gustke N, Mandelkow E (1993) Microtubule-associatedprotein tau, paired helical filaments, and phosphorylation. Ann N YAcad Sci 695:209–216.

euer K, Suppanz IE, Lingor P, Planchamp V, Goricke B, Fichtner L,Braus GH, Dietz GP, Jakobs S, Bahr M, Weishaupt JH (2007)Cyclin-dependent kinase 5 is an upstream regulator of mitochon-drial fission during neuronal apoptosis. Cell Death Differ 14:651–661.

unoz JP, Alvarez A, Maccioni RB (2000) Increase in the expression ofthe neuronal cyclin-dependent protein kinase cdk-5 during differenti-ation of N2A neuroblastoma cells. Neuroreport 11:2733–2738.

oble W, Olm V, Takata K, Casey E, Mary O, Meyerson J, Gaynor K,LaFrancois J, Wang L, Kondo T, Davies P, Burns M, Veeranna,Nixon R, Dickson D, Matsuoka Y, Ahlijanian M, Lau LF, Duff K(2003) Cdk5 is a key factor in tau aggregation and tangle formation

in vivo. Neuron 38:555–565.

areek TK, Kulkarni AB (2006) Cdk5: a new player in pain signaling.Cell Cycle 5:585–588.

atrick GN, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai LH(1999) Conversion of p35 to p25 deregulates Cdk5 activity andpromotes neurodegeneration. Nature 402:615–622.

atzke H, Maddineni U, Ayala R, Morabito M, Volker J, Dikkes P,Ahlijanian MK, Tsai LH (2003) Partial rescue of the p35-/- brainphenotype by low expression of a neuronal-specific enolase p25transgene. J Neurosci 23:2769–2778.

orter AG, Jänicke RU (1999) Emerging roles of caspase-3 in apo-ptosis. Cell Death Differ 6:99–104.

chneider A, Biernat J, von Bergen M, Mandelkow E, Mandelkow EM(1999) Phosphorylation that detaches tau protein from microtu-bules (Ser262, Ser214) also protects it against aggregation intoAlzheimer paired helical filaments. Biochemistry 38:3549–3558.

elkoe DJ (1989) Biochemistry of altered brain proteins in Alzheimer’sdisease. Annu Rev Neurosci 12:463–490.

akahashi S, Ohshima T, Cho A, Sreenath T, Iadarola MJ, Pant HC,Kim Y, Nairn AC, Brady RO, Greengard P, Kulkarni AB (2005)Increased activity of cyclin-dependent kinase 5 leads to attenua-tion of cocaine-mediated dopamine signaling. Proc Natl Acad SciU S A 102:1737–1742.

sai LH, Delalle I, Caviness VS Jr, Chae T, Harlow E (1994) p35 Is aneural-specific regulatory subunit of cyclin-dependent kinase 5.Nature 371:419–423.

seng HC, Zhou Y, Shen Y, Tsai LH (2002) A survey of Cdk5 activatorp35 and p25 levels in Alzheimer’s disease brains. FEBS Lett523:58–62.

treras E, Futatsugi A, Rudrabhatla P, Keller J, Iadarola MJ, Pant HC,Kulkarni AB (2009) Tumor necrosis factor-alpha regulates cyclin-dependent kinase 5 activity during pain signaling through transcrip-tional activation of p35. J Biol Chem 284:2275–2284.

eishaupt JH, Neusch C, Bahr M (2003) Cyclin-dependent kinase 5(CDK5) and neuronal cell death. Cell Tissue Res 312:1–8.

heng YL, Kesavapany S, Gravell M, Hamilton RS, Schubert M, AminN, Albers W, Grant P, Pant HC (2005) A Cdk5 inhibitory peptidereduces tau hyperphosphorylation and apoptosis in neurons.

EMBO J 24:209–220.

(Accepted 2 April 2009)(Available online 9 April 2009)