Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase...

Expression Pattern of Ataxia TelangiectasiaMutated (ATM), p53, Akt, and GlycogenSynthase Kinase-3b in the Striatum of RatsTreated With 3-Nitropropionic Acid

Joaquim Duran-Vilaregut,1,2 Gemma Manich,1 Jaume del Valle,1,2

Antoni Camins,2,3 Merce Pallas,2,3 Jordi Vilaplana,1,2 and Carme Pelegrı1,2*1Departament de Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain2Centros de Biomedicina en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain3Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Institut de Biomedicina (IBUB),Universitat de Barcelona, Barcelona, Spain

3-Nitropropionic acid (3-NPA) is a mitochondrial toxinused in the laboratory to replicate neurodegenerativeconditions that are accompanied by degeneration ofthe caudate-putamen. 3-NPA induces depletion in ATPproduction, reactive oxygen species production, andsecondary excitotoxicity mediated by activation ofN-methyl-D-aspartate receptors that culminates in thetriggering of cell death mechanisms, including apopto-sis. We here examined by immunohistochemicalmethods whether cellular expression of phosphoSer1981-ataxia telangiectasia mutated (ATM), phosphoSer15-p53,phosphoSer473-Akt, and phosphoSer9-glycogen synthasekinase-3b (GSK3b), which are key signal moleculesthat play a critical role in regulating cellular processesrelated to cell survival and demise, were involved in thestriatal neurodegeneration in the brains of rats treatedwith 3-NPA. Our results indicate that the toxin inducedthe activation of ATM and p53 only in astrocytes, and arole for these proteins in neuronal degeneration wasruled out. On the other hand, striatal neurons lost theactive form of Akt as soon as they began to appearpyknotic, indicating impairment of the PI3K/Akt/GSK3pathway in their degenerative process. The inactiveform of GSK3b was detected extensively, mainly in therim of the striatal lesions around degenerating neurons,which could be attributed to a cell death or cell survivalresponse. VVC 2012 Wiley Periodicals, Inc.

Key words: neurotoxin; immunohistochemistry; rat

3-Nitropropionic acid (3-NPA) is a mitochondrialtoxin and its precursor, 3-nitropropanol, is present inplants such as Astragalus and fungus such as Arthrinium(Ludolph et al., 1991). In humans, 3-NPA producespreferential degeneration of the putamen and caudatenucleus, associated with severe neurological symptoms.Systemic administration of the toxin to experimental ani-mals such as nonhuman primates or rodents producessimilar effects. Thus, 3-NPA is used in the laboratory to

replicate neurodegenerative conditions associated withdegeneration of the caudate-putamen (Brouillet et al.,2005). 3-NPA has been used mainly to study Hunting-ton’s disease (HD), a neurodegenerative disorder prefer-entially affecting the striatum, in which deficits in energymetabolism and dysfunction of mitochondrial chaincomplexes (MCCs) play a pivotal role (Gu et al., 1996;Browne et al., 1997). In addition, 3-NPA is suitable formodeling features of acute neuronal insults, such as cere-bral ischemia (Pang et al., 2003). The toxin irreversiblyinhibits succinate dehydrogenase (SDH), the main con-stituent of the MCC II (Brouillet et al., 1999). A recentstudy (Liot et al., 2009), performed in cultured corticalneurons, revealed that 3-NPA-mediated inhibition ofSDH causes an initial dramatic depletion in ATP pro-duction. This depletion is concomitant to a mild increasein reactive oxygen species (ROS) and delayed secondaryexcitotoxicity, mediated by activation of N-methyl-D-aspartate receptors (NMDAR) by glutamate. This activa-tion in turn induces a second rise in ROS productionand mitochondrial fragmentation, which triggers neuro-nal cell death.

Contract grant sponsor: Ministerio de Educacion y Ciencia; Contract

grant numbers: BFU/2009-08352, BFU/2010-22149, SAF/2009-13093;

Contract grant sponsor: Instituto de Salud Carlos III (FEDERFOUNDS);

Contract grant number: PI080400; Contract grant sponsor: Programa

Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED);

Contract grant number: 610RT0405; Contract grant sponsor: Generalitat

de Catalunya; Contract grant number: 2009/SGR853.

J. Vilaplana and C. Pelegrı contributed equally to this work.

*Correspondence to: Carme Pelegrı, Departament de Fisiologia, Facultat

de Farmacia, Av. Joan XXIII s/n, E-08028 Barcelona, Spain.

E-mail: [email protected]

Received 21 December 2011; Revised 16 February 2012; Accepted 4

March 2012

Published online in Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/jnr.23060

Journal of Neuroscience Research 00:000–000 (2012)

' 2012 Wiley Periodicals, Inc.

Both necrosis and apoptosis, which have tradition-ally been considered as the basic mechanisms of celldeath, are involved in 3-NPA-mediated neurotoxicity invitro (Pang and Geddes, 1997; Nasr et al., 2003; Panget al., 2003). With in vivo administration, it has beenreported that calpain, a cysteine protease that is activatedby Ca21 and is related to necrotic processes (Wang,2000), is a predominant effector of striatal cell deathwhen SDH is persistently inhibited by chronic adminis-tration of 3-NPA (Bizat et al., 2003a,b; Galas et al.,2004). In contrast, transient inhibition of SDH by acuteor subacute treatment with the toxin leads to the addi-tional activation of caspase-3, the main effector of theapoptotic process, as has been shown in our laboratory(Duran-Vilaregut et al., 2010) and others (Duan et al.,2000; Bizat et al., 2003a).

Several signaling pathways could be responsible foractivation of the apoptotic process that culminates in cas-pase-3 activation and subsequent neuronal demise afteracute or subacute 3-NPA administration. It has been pro-posed that the toxin induces overproduction of ROS,which causes oxidative DNA damage and DNA fragmen-tation (Kim and Chan, 2002). Thus, we first focused onthe study of the activation of ataxia telangiectasia mutated(ATM), which is activated by autophosphorylation atSer1981 in response to DNA damage signals. It thenphosphorylates a number of proteins that are essential tothe control of cell cycle checkpoints; DNA repair; and, inthe case of excessive DNA damage, cell death(McKinnon, 2004). In addition to ATM, we studied theform of p53 phosphorylated at Ser15, one of the mainsubstrates of the former (Shiloh, 2003). p53 Is a transcrip-tion factor that is activated in response to stress signals andelicits several cellular responses, including growth arrest,senescence, and apoptosis (Polager and Ginsberg, 2009).On the other hand, it has been reported that an increasein the phosphorylated form of Akt is protective against 3-NPA-induced neurodegeneration (Lee et al., 2008a,b;Almeida et al., 2009). Akt, also called protein kinase B, is aserine/threonine protein kinase of the PI3K pathway thatplays a central role in cell survival, in that it blocks thefunction of proapoptotic proteins and processes (Manningand Cantley, 2007). Here we studied its fully activatedform, which requires phosphorylation at Ser473 (Alesiet al., 1999; Liao and Hung, 2010). Moreover, we ana-lyzed the b isoform of glycogen synthase kinase-3(GSK3) phosphorylated at Ser9, which is a main substrateof Akt (Cohen and Frame, 2001). GSK3 is a serine/thre-onine protein kinase with two isoforms, namely, GSK3aand GSK3b, which was initially described as a keyenzyme in glucogen metabolism, although recent evi-dence indicates that it can modulate apoptosis in a widerange of neurotoxic conditions (Jope and Johnson, 2004).

MATERIALS AND METHODS

Animals and 3-NPA Treatment

Male Sprague-Dawley rats (220–250 g; Harlan Inter-fauna Iberica, Barcelona, Spain) had access to food and water

ad libitum and were kept under standard conditions of tem-perature (228C 6 28C) and on 12:12-hr light–dark cycles(300 lx/0 lx). Studies were performed in accordance with theinstitutional guidelines for the care and use of laboratory ani-mals established by the Ethical Committee for Animal Experi-mentation at the University of Barcelona. One group of 10rats (treated group) was injected with saline solution contain-ing 3-NPA (Sigma-Aldrich, St. Louis, MO) adjusted to pH7.4 with NaOH at a dose of 20 mg/kg i.p. once per day for3 days (days 0, 1, and 2). The control group, which includedfive animals, was injected only with saline solution. On day 6,both groups of animals were killed as described below, andtheir brains were excised. All rats were evaluated daily fromday 0 until day 6 of experiment for both body weight lossand motor impairment, and the evolution observed was simi-lar to that described from previous studies performed in ourlaboratory (Duran-Vilaregut et al., 2009).

Brain Processing

Brains for immunohistochemistry and TUNEL stainingwere obtained as follows. Rats were anesthetized with 80 mg/kg i.p. sodium pentobarbital and intracardially perfused with100 ml physiological saline. Brains were then excised and snapfrozen by immersion in isopentane chilled in dry ice andstored at 2808C. Thereafter, brains were embedded in OCTcryostat-embedding compound (Tissue-Tek, Torrance, CA),and 20-lm-thick cryostatic sections containing striatum wereobtained. Slices were picked up on common slides, fixed withacetone for 10 min at 48C, allowed to dry at room tempera-ture, and finally frozen at 2208C until staining.

Immunohistochemistry of Brain Sections

Slides with brain sections were allowed to thaw at roomtemperature and then rehydrated with PBS for 5 min beforebeing blocked and permeabilized with PBS containing 1%bovine serum albumin (Sigma-Aldrich) and 0.1% TritonX-100 (Sigma-Aldrich) for 20 min. After two 5-min washesin PBS, slides were incubated with the primary antibody (seebelow) for 90 min at room temperature. They were thenwashed again and incubated for 1 hr at room temperature inthe dark with the secondary antibody (see below). Fiveminutes before the end of the second incubation, nuclearstaining was performed by adding Hoechst (H-33258; Sigma-Aldrich) reagent at 10 lg/ml in PBS to the incubationmedium to a final concentration of 2 lg/ml. Finally, slideswere washed, mounted using Prolong Gold (Invitrogen,Carlsbad, CA) antifade medium, allowed to dry overnight atroom temperature, and stored at 48C in the dark. Stainingcontrols were performed by incubating with PBS instead ofthe primary antibody or instead of both antibodies.

The following primary antibodies were used: rabbit pol-yclonal antiphosphoSer1981-ATM (R&D Systems, Minneapolis,MN; dilution 1:200), rabbit polyclonal antiphosphoSer15-p53(R&D Systems; dilution 1:100), rabbit polyclonal antiphos-phoSer9-GSK3b (Cell Signaling, Danvers, MA; dilution1:100), rabbit polyclonal antiphosphoSer473-Akt (Abcam, Cam-bridge, United Kingdom; dilution 1:100), mouse monoclonalanti-NeuN (Millipore, Billerica, MA; dilution 1:100), mouse

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Journal of Neuroscience Research

monoclonal anti-GFAP (Neomarkers, Fremont, CA; dilution1:100), and chicken polyclonal anti-GFAP (Millipore; dilution1:100). AlexaFluor 488 donkey anti-mouse IgG, AlexaFluor488 anti-chicken IgG, AlexaFluor 555 donkey anti-rabbitIgG, and AlexaFluor 660 anti-mouse IgG (Invitrogen; dilution1:250) were used as secondary antibodies.

Fluorescence Microscopy Analysis

Brain sections from 3-NPA-treated and control rats wereexamined under a laser confocal microscope (TCS/SP2; LeicaMicrosystems, Wetzlar, Germany). In some cases, series of con-secutive images were obtained for each staining and experimen-tal group. In the case of treated animals, the sequence of imagesincluded injured and noninjured areas of the striatum. Oncethe image series had been collected, the images were rebuilt inorder to obtain single images which, subsequently, weremerged in Image J (National Institutes of Mental Health) andPhotoshop (Adobe Systems, San Jose, CA). The authors declarethat there are no conflicts of interest.

RESULTS

Description of the Striatal Lesion Inducedby 3-NPA

According to previous studies (Duran-Vilaregutet al., 2009, 2010), we defined three striatal regions inour studies with the 3-NPA model (Fig. 1). The regioncalled ‘‘noninjured striatum’’ included all the striatal areasin which neurons and astrocytes (stained with thechicken polyclonal antiglial fibrillary acidic protein(GFAP) antibody) were not affected by the treatmentwith the toxin, so they did not show morphologicalalterations compared with nontreated rats. A magnifica-tion of this area is included in Figure 1A, in whichastrocytes and neurons unaffected by 3-NPA can beobserved. The ‘‘perilesional rim’’ included the astroglio-sis area and the outer area of the striatal lesion, where,according to our previous study (Duran-Vilaregut et al.,

2010), apoptotic neurons positive for active caspase-3and TUNEL stains were mainly found. In the astrogliosisarea, astrocytes appeared in their reactive form (Fig. 1B),showing stronger GFAP staining and more processesthan astrocytes in the noninjured striatal area. In theouter area of the striatal lesion, neurons started to showa pyknotic appearance (Fig. 1C), and, although astroglio-sis had been lost, residual GFAP staining was stilldetected. Reactive astrocytes in contact with the outerarea of the lesion showed altered morphology, indicatingthat they experienced a degeneration process. Finally, inthe ‘‘lesion core’’ region, neurons were pyknotic (Fig.1D). According to our previous study (Duran-Vilaregutet al., 2010), they barely showed active caspase-3 orTUNEL stains. Residual GFAP staining was also foundin this region.

Although the size and extent of the lesion weredifferent in each animal, the different striatal studiedareas could be observed in all of them. In each area, thecellular pattern and the protein expression were equiva-lent in all animals.

Expression Pattern of PhosphoSer1981-ATMand PhosphoSer15-p53

Figure 2 shows representative images correspondingto striatal areas of brains from control and 3-NPA-treated rats immunostained with an antibody that recog-nizes the form of ATM phosphorylated at Ser1981. Asshown in Figure 2A, the striatum of control animals didnot show any staining for phosphoSer1981-ATM. In 3-NPA-treated animals, noninjured areas of their striatumhad the same appearance as controls (Fig. 2B). The coreof the striatal lesion did not show any positive stainingfor the kinase (Fig. 2C). Positive staining for phos-phoSer1981-ATM was detected only on the rim of thestriatal lesion, where tissue had started to lose its charac-teristic structure (Fig. 2D1,E1). Subsequently, phos-phoSer1981-ATM staining was combined with NeuN

Fig. 1. Staining of neurons and astrocytes in the striatum of 3-NPA-treated rats. Representative image of the striatum of a rat from the 3-NPA-treated group immunostained with the anti-NeuN antibody andthe anti-GFAP antibody, including the three regions studied. Magnifi-cations of each of these regions are shown: noninjured striatum (A),perilesional rim (B,C), and lesion core (D). Solid arrow indicates

astrocytes not affected by 3-NPA. Solid arrowheads indicate neuronsnot affected by 3-NPA. Open arrows indicate reactive astrocytes.Open arrowheads indicate pyknotic neurons in the lesion induced by3-NPA. Small arrow indicates residual GFAP staining in the lesioninduced by 3-NPA. Scale bars 5 50 lm. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]

ATM and Akt in 3-NPA Neurodegeneration 3


Fig. 2. PhosphoSer1981-ATM staining is detected on the rim of thestriatal lesion of 3-NPA-treated rats. Representative images of thestriatum of control (A) and 3-NPA-treated (B,C,D1,E1) rats immu-nostained with the anti-phosphoSer1981-ATM antibody. In the case ofthe rim of the striatal lesion of 3-NPA-treated animals, magnifica-tions in which phosphoSer1981-ATM staining overlaps with NeuN

(D2) and GFAP (E2) staining are included. Insets in D3 and E3show a magnification of the selected area that includes Hoechststaining. ATM-P, phosphoSer1981-ATM. Scale bars 5 50 lm. [Colorfigure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]



staining to investigate whether cells expressing the activekinase were neurons. Figure 2D3 shows that phos-phoSer1981-ATM was present in the area where neuronsbegan to appear pyknotic, although they did not expressit in any case. Thus, we next tested whether phos-phoSer1981-ATM was expressed by astrocytes. Figure 2E3shows that phosphoSer1981-ATM staining was detected inthe nuclei of the reactive astrocytes (stained with themouse monoclonal anti-GFAP antibody) located in theastrogliosis area surrounding the lesion. Insets in Figure2D3,E3 show that phosphoSer1981-ATM staining was nu-clear, insofar as it colocalized with Hoechst staining.Because GFAP stains the processes of astrocytes, colocal-ization of GFAP and phosphoSer1981-ATM was notvisible.

We next studied activation of one of the main tar-gets of ATM, p53. Figure 3 shows representative imagesof staining of p53 phosphorylated at Ser15, in striatafrom control and 3-NPA-treated rats. PhosphoSer15-p53was not detected either in control animals or in the non-injured striatal areas of 3-NPA-treated animals (Fig.3A,B, respectively). However, in contrast to ATM, posi-tive staining for p53 was seen both on the rim (Fig.3F1) and in the core (Fig. 3C,D1,E1) of the striatallesion of 3-NPA-treated rats. Combination of phos-phoSer15-p53 and NeuN stains showed that the proteinwas not expressed by neurons (Fig. 3D3). In contrast,double staining with the chicken polyclonal anti-GFAPantibody showed that, as for ATM, phosphoSer15-p53was expressed in astroglia (Fig. 3E3,F3). In the core ofthe lesion, p53 was found mainly in accumulations ofresidual GFAP staining that had completely lost thecharacteristic appearance of astrocytes (Fig. 3E3),whereas, on the rim surrounding the lesion, it wasdetected in degenerating astrocytes (Fig. 3F3). Insets inFigure 3D3,E3,F3 show that phosphoSer15-p53 stainingwas preferentially detected next to Hoechst staining butdid not colocalize.

Expression Pattern of PhosphoSer473-Aktand PhosphoSer9-GSK3b

Figure 4 shows representative images of striatal areasfrom control and 3-NPA-treated rats immunostained withan antibody directed against Akt phosphorylated atSer473. As shown in Figure 4A3, phosphoSer473-Akt wasconstitutively expressed in striatal neurons from controlrats. Insets in Figure 4A1–A3 show that its expression wasnuclear. In 3-NPA-treated animals, phosphoSer473-Aktwas also present in neurons located in the noninjuredareas of the striata (Fig. 4B3, noninjured area). In contrast,degenerating neurons in the injured area did not showany staining for the kinase (Fig. 4B3, perilesional rim andlesion core).

Figure 5 shows representative images of striatalareas from control and 3-NPA-treated rats immuno-stained with an antibody that specifically recognizesphosphoSer9-GSK3b. In the injured striatal area oftreated animals, phosphoSer9-GSK3b staining was

strongly detected on the rim of the lesion in the formof dense clumps (Fig. 5B1, rim of the lesion). Stainingwas weaker closer to the core (Fig. 5B1, lesion core).Colocalization with NeuN staining showed that phos-phoSer9-GSK3b staining on the rim of the lesion waspreferentially found in pyknotic neurons, and it seemedto be perikaryal (Fig. 5B3, arrowheads). Toward thecore of the lesion, pyknotic neurons with perikaryalphosphoSer9-GSK3b staining were barely detected(Fig. 5B3, arrows).

DISCUSSION

Expression Pattern of PhosphoSer1981-ATM andPhosphoSer15-p53.

Our results indicate that 3-NPA treatment did notinduce phosphorylation of ATM and p53 in neurons ofthe striatum of intoxicated rats. In contrast, the activeform of ATM, phosphoSer1981-ATM, was detected inreactive astrocytes in the area of astrogliosis that sur-rounded the 3-NPA-induced lesion. PhosphoSer15-p53,one of the main products of active ATM, was alsodetected only in association with degenerating astrocyteslocated both in the core and on the rim of the lesion.ATM is activated in response to double strand breaks inDNA, and it then phosphorylates substrates involved inrecognizing DNA damage or in signaling this damage tocell-cycle checkpoints (Shiloh, 2003; Barzilai et al.,2008). Kruman et al. (2004) showed that, in culturedneurons, exposure to genotoxic compounds inducesDNA damage that triggers cell cycle re-entry and subse-quent apoptosis and that pretreatment with ATM inhibi-tors such as caffeine or wortmannin abrogates bothevents without affecting the DNA damage. These find-ings pointed to a close relationship among DNA dam-age-induced apoptosis, activation of ATM, and cell cyclere-entry in postmitotic neurons and supported previousstudies in which neurons from ATM-deficient mice hadbeen shown to be resistant to DNA damage-induced ap-optosis (Chong et al., 2000; Lee and McKinnon, 2000;Macleod et al., 2003). 3-NPA treatment inducesoverproduction of ROS, which causes oxidative DNAdamage and DNA fragmentation, which were detectedby 8-hydroxyguanosine immunohistochemistry andTUNEL staining, respectively (Kim and Chan, 2002).However, the lack of neuronal ATM activation shownhere suggests that 3-NPA neuronal toxicity is not medi-ated by genotoxic effects. In a previous study performedwith our 3-NPA experimental model, TUNEL stainingwas confined to the striatal lesion, mainly on its rim, andwas widely detected in neurons that expressed active cas-pase-3 (Duran-Vilaregut et al., 2010), indicating thatDNA fragmentation could be the final stage of the apo-ptotic cascade rather than a manifestation of the geno-toxic effect of 3-NPA. In support of this, it has beenreported that 3-NPA induces specific mitochondrialDNA damage and does not alter nuclear DNA (Manda-villi et al., 2005).



Fig. 3. PhosphoSer15-p53 staining is detected on the rim and core ofthe striatal lesion of 3-NPA-treated rats. Representative images of thestriatum of control (A) and 3-NPA-treated (B,C,D1,E1,F1) ratsimmunostained with the anti-phosphoSer15-p53 antibody are shown.In the case of 3-NPA-treated animals, magnifications in which phos-phoSer15-p53 overlaps with NeuN staining in the core of the lesion

(D2) and with GFAP staining in the core (E2) and on the rim (F2) ofthe striatal lesion are included. Insets in D3, E3, and F3 show a mag-nification of the selected area that includes Hoechst staining. p53-P,phosphoSer15-p53. Scale bars 5 50 lm. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]


On the other hand, administration of 3-NPAinduced the activation of ATM in reactive astrocyteslocated in the area of astrogliosis that surrounded thestriatal lesion of intoxicated rats. Astrocytes interactclosely with surrounding neurons by providing themwith energy and substrates for neurotransmission, pro-tecting and modulating their growth and synaptic func-tion (Travis, 1994; Allen and Barres, 2009). Neurons areparticularly vulnerable to oxidative stress, and they relyheavily on metabolic coupling with astrocytes for antiox-idant support, since the latter have stronger antioxidant

potential (Shih et al., 2003; Liu et al., 2005). It has beenreported that Atm2/2 astrocytes show growth abnormal-ities and increased oxidative stress (Liu et al., 2005).Moreover, the absence of ATM results in oxidative stressin the brain (Kamsler et al., 2001; Chen et al., 2003).These observations indicate that ATM may modulate in-tracellular redox status. Recently, it has been shown thatactivation of ATM induced by oxidative stress can occurin the absence of DNA damage (Guo et al., 2010).According to this evidence, activation of ATM in reac-tive astrocytes on the rim of the striatal lesion may be a

Fig. 4. PhosphoSer473-Akt is lost in degenerating neurons in the striatallesion of 3-NPA-treated rats. Representative images of the striatum ofcontrol (A1–3) and 3-NPA-treated (B1–3) rats immunostained withthe antiphosphoSer473-Akt and anti-NeuN antibodies. In the case of 3-NPA-treated animals, images were obtained by overlapping serialphotographs from the striatum including noninjured and injured areas.

A1,B1: Staining of phosphoSer473-Akt. A2,B2: Neurons stained withthe anti-NeuN antibody. A3,B3: Overlapping of phosphoSer473-Aktand NeuN staining. Insets in A1–3 show a magnification of theselected area that includes Hoechst staining. AKT-P, phosphoSer473-Akt. Scale bars 5 50 lm. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]



mechanism involved in re-establishing redox status andprotecting neurons against the oxidative insult inducedby 3-NPA administration.

Expression Pattern of PhosphoSer473-Akt andPhosphoSer9-GSK3b

Our results show that the activated form of Akt,which is phosphorylated at Ser473, is constitutively

expressed by neurons in healthy striatal tissue from con-trol and 3-NPA-treated rats, although it is completelylost in pyknotic neurons located in the lesioned area ofthe striatum in intoxicated animals. Akt is a central nodein cell signaling downstream of growth factors, cyto-kines, and other cellular stimuli. It plays a critical role incell survival by inhibiting, through phosphorylation, thefunction of proapoptotic proteins such as BAD and

Fig. 5. PhosphoSer9-GSK3b is strongly detected on the rim of thestriatal lesion of 3-NPA-treated rats. Representative images of thestriatum of control (A1–3) and 3-NPA-treated (B1–3) rats immuno-stained with the antiphosphoSer9-GSK3b and anti-NeuN antibodies.In the case of 3-NPA-treated animals, images were obtained byoverlapping serial photographs from the striatum including nonin-jured and injured areas. A1,B1: Staining of phosphoSer9-GSK3b.

A2,B2: Neurons stained with the anti-NeuN antibody. A3,B3:Overlapping of phosphoSer9-GSK3b and NeuN staining. Arrowheadsshow pyknotic neurons with perikaryal GSK3b staining clumps inthe rim of the lesion. Arrows indicate pyknotic neurons with peryka-rial GSK3b staining in the core of the lesion. GSK3b-P, phos-phoSer9-GSK3b. Scale bars 5 50 lm. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]



caspase-9 and transcription factors such as FOXO pro-teins. These, in turn, promote transcription of otherapoptotic proteins such as BIM and Fas ligand whenactive (Manning and Cantley, 2007). Consistently withour findings, it has been reported in rats that concomi-tant administration of atorvastain (Lee et al., 2008a) orgranulocyte colony-stimulating factor (Lee et al., 2008b)with 3-NPA protects against the striatal neurodegenera-tion induced by the toxin. Moreover, in an in vitrostudy, brain-derived neurotrophic factor prevented mito-chondrial-dependent apoptosis induced by 3-NPA byreducing caspase-3 activation and chromatin condensa-tion/fragmentation (Almeida et al., 2009). In all threestudies, the protective treatment induced an increase inthe phosphorylated form of Akt, indicating that its lossmight be involved in 3-NPA-induced neurodegenera-tion. Furthermore, administration to rats of lentiviralvectors expressing the first 171 N-terminal aminoacids of huntingtin with a pathological polyglutaminesequence of 82 units induces striatal HD neuropathologyaccompanied by downregulation of total and phos-phoSer473-Akt levels in neurons before they degenerate(Colin et al., 2005). This indicates that the decrease wasnot merely a consequence of apoptosis but a mechanisminvolved in it. Remarkably, the same study detected, inHD post-mortem brains, high levels of a truncated andinactive form of Akt resulting from caspase-3 cleavage,particularly in the striata. This confirms the involvementof the protease in the development of the disease, as the3-NPA model also suggests (Duan et al., 2000; Duran-Vilaregut et al., 2010). A marked decrease in total andphosphoSer473-Akt has also been detected in degeneratingdopaminergic neurons from substantia nigra pars com-pacta in post-mortem brains of Parkinson’s diseasepatients (Timmons et al., 2009). This finding has beenreproduced in models of the disease in vitro, induced bytoxins such as 6-hydroxidopamine (Malagelada et al.,2008). Likewise, senescence accelerated prone 10(SAMP10), a mouse model of early-onset neurodegener-ative dementia diseases, showed reduced hippocampallevels of phosphoSer473-Akt after 6 months of age, whenmice showed obvious deterioration in performance oflearning and memory tasks (Nie et al., 2009). Thus, ourresults are consistent with a growing body of evidencepointing to impairment of Akt signaling pathway as akey event in neuronal degeneration.

With regard to GSK3b, our results indicate astrong increase in the expression of the inhibited form ofthe kinase phosphorylated in Ser9 in the striatal lesion of3-NPA-treated rats compared with control animals.PhosphoSer9-GSK3b was found mainly forming denseperikaryal neuronal clumps, on the rim of the striatallesion, where the majority of active caspase-3- and/orTUNEL-positive neurons were detected after 3-NPAtreatment (Duran-Vilaregut et al., 2010). GSK3 is akinase that, although traditionally associated exclusivelywith the metabolism of glycogen, is now recognized as akey regulator of numerous signaling pathways. Whendysregulated, such pathways have been implicated in the

development of neurodegenerative diseases such as Alz-heimer’s disease, among others (Doble and Woodgett,2003; Jope and Johnson, 2004). Paradoxical findingshave been reported regarding the involvement of GSK3in apoptosis. On the one hand, it has been shown thatits aberrant activation promotes apoptosis in a wide vari-ety of paradigms of cell death, which include toxicityinduced by amyloid-b peptide and mitochondrial toxins(Grimes and Jope, 2001) and HD-associated polyglut-amine toxicity (Carmichael et al., 2002). On the otherhand, transgenic mice in which GSK3b activity wasinhibited through the expression of a dominant-negativeform of the protein showed increased neuronal apopto-sis, which preferentially affected the striatum (Gomez-Sintes et al., 2007; Gomez-Sintes and Lucas, 2010).These apparent contradictory findings are the result ofGSK3 having opposite effects on the two major apopto-tic signaling pathways. It has been established that thekinase, when active, promotes the intrinsic apoptoticpathway, by facilitating the function of proapoptoticproteins, but it inhibits the extrinsic pathway by pre-venting the activation of the initiator caspase-8 (Beureland Jope, 2006). According to this line of evidence, ifthe death receptor-mediated extrinsic pathway of apo-ptosis is activated in the striatal lesion of 3-NPA-treatedrats, then the inactivation of GSK3b that we found heremay contribute to its progression, because activation ofcaspase-8 would not be inhibited. This would be con-sistent with the fact that sustained GSK3 inhibition pref-erentially increases apoptosis in the striatum, as has beenreported by Gomez-Sintes et al. (2007).

Given that the active form of Akt is absent indegenerating neurons located in the striatal lesion of 3-NPA-treated rats, other kinases may be involved in theinactivation of GSK3. In addition to Akt, several kinases,including protein kinase A (PKA) and protein kinase C(PKC), can phosphorylate GSK3 in Ser9 (Doble andWoodgett, 2003), in a mechanism that has been relatedto the promotion of survival of neurons in both cases.cAMP-mediated activation of PKA and subsequent inac-tivation of GSK3b promoted survival in cerebellar gran-ule neurons subjected to potassium deprivation inde-pendently of Akt activation (Li et al., 2000). Moreover,recent findings showed that activation of PKC throughNMDAR-mediated extracellular calcium entry induceda survival response in cerebellar granule neurons that wasnot suppressed by PI3K/Akt pathway impairment(Ortega et al., 2010). The latter is consistent with thefact that two types of NMDAR are present in neurons,NR2A and NR2B, the activation of which has beenlinked to the triggering of prosurvival and proapoptoticmechanisms, respectively (Broughton et al., 2009).According to this view, because excitotoxicity mediatedby NMDAR activation and cytosolic calcium increase isa key mechanism in 3-NPA-mediated neurotoxicity(Liot et al., 2009), inactivation of GSK3b on the rimof the striatal lesion of 3-NPA-treated rats may be asurvival response induced by prosurvival NMDAR-mediated PKC activation.



CONCLUSIONS

The aim of this study was to establish whetherphosphorylation of ATM, p53, Akt, and GSK3b, whichare key signal molecules related to cellular survival anddemise processes, were involved in neuronal apoptosisdegeneration in the striatal lesion induced by 3-NPAadministration. According to our findings, the toxininduced the activation of ATM and phosphorylation ofits main target p53 in astrocytes but not in neurons. Thiswould tend to rule out the involvement of this signalingpathway in 3-NPA-induced neuronal degeneration.With respect to Akt and GSK3b, neurons lost nuclearphosphoSer473-Akt as soon as they started to appearpyknotic, which suggests that the loss of this kinase has adetermining role in their degeneration. Although theactive form of Akt was lost, the form of GSK3b phos-phorylated in Ser9 was increased and extensivelydetected in the striatal lesion of 3-NPA-treated rats,mainly on the rim, in some of the pyknotic neurons.More studies are required to establish whether inactiva-tion of GSK3b in the striatal lesion of 3-NPA-treatedrats is more likely to contribute to neurodegenerationthrough the activation of the extrinsic apoptotic pathwayor to be part of a survival response against neuronal apo-ptosis induced by the toxin.

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Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase...

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