Three major forms of cell death, apoptosis, necrosisand autophagic/vacuolar cell death, are well character-ized (8). Necrosis is known to be initiated accidentally.The necrotic stimuli induce vacuolation of cytoplasm,breakdown of plasma membrane, release of cellularcontents and pro-inflammatory molecules. Recently, aprogrammed necrotic death called autophagy has beenidentified. Autophagy contains both necrosis and apo-ptosis characteristics (8, 19). Among these three typesof cell death, apoptosis has got the most attention. It isan active physiological process of cellular self destruc-tion which regulates development and tissue homeosta-sis (38). This suicidal program is composed of twomajor phases, commitment and execution, which areactivated by external stimuli via ligand-receptor interac-

tion or by internal stimuli (6). This stimulation acti-vates the recruitment of several signal transduction fac-tors, including initiator caspases and the pro-apoptotic-bcl-2 family (11). Transduction of these pro-apoptoticstimuli via different signaling pathways results in eitherthe depolarization of mitochondrial membrane poten-tial, the release of cytochrome c from the mitochondrialintermembrane space to the cytoplasm or the directactivation of caspases. The execution phase includesthe apoptosome-mediated procaspase activation, whichin turn stimulates the downstream effector caspases(caspase-3, -6 and -7) (1, 34). The activation of thesegene cascades indeed leads to cell shrinkage, nuclearcondensation, degradation of genomic DNA, membraneredistribution of phospholipid and ultimately to celldeath.

Screening of Pro-Apoptotic Genes Upregulated inan Experimental Street Rabies Virus-InfectedNeonatal Mouse Brain

Sukathida Ubol*, 1, Jitra Kasisith1, Dhanesh Pitidhammabhorn1, and Veera Tepsumethanol2

1Department of Microbiology, Faculty of Science, Mahidol University, 272 Rama VI Rd., Ratchatewee, Bangkok 10400, Thai-land, and 2Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

Received January 31, 2005; in revised form, March 7, 2005. Accepted March 10, 2005

Abstract: Rabies virus (RABV) is able to induce apoptotic death of target cells. The molecular pathway ofRABV-induced cell death is partially known. In the present study, cDNA array analysis was used as a toolto screen for pro-apoptotic genes that may be involved in RABV induction. RNA was extracted from theinfected CNS and from mock-infected controls. When the mean gene expression was compared betweenthe infected group and controls, 21 potential apoptotic genes were identified that exhibited more than 2.5-fold difference in their expression levels. These 21 genes can be grouped into two groups, those genes thatparticipate in the commitment phase and those that play a role as executioners. Examples of genes in com-mitment phase were death receptors (Fas-L receptor, TNF-receptor), lysosomal proteases, calpain, caspase-1, signaling molecules (ERK, p38MAPK) and bcl-2 family members. Cytochrome c and caspase-3 wererepresentatives of executioners. Based on types of genes activated during the commitment phase, twoindependent apoptotic mechanisms may be activated in response to the RV infection. The first is immune-mediated death which may operate through the receptor-ligand pathway activated by caspase-1 and thepro-inflammatory cytokine, IL-1�. The other mechanism is a protease-mediated process which involveslysosomal proteases and calcium-dependent neutral proteases. These two stimulating pathways were fol-lowed by Bad, Bak, Bid activation and subsequently the upregulation of cytochrome c and caspase-3. Inaddition, mobilization of K� ion and other accessory apoptotic genes such as annexins and clusterin werealso upregulated.

Key words: Rabies virus, Apoptosis, Caspases, Calpain, Lysosomal proteases

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Microbiol. Immunol., 49(5), 423–431, 2005

Abbreviations: Ca2�, calcium ion; Cl�, chloride ion; IL-1β,interleukin-1-beta; IL-6, interleukin-6; K�, potassium ion.

*Address correspondence to Dr. Sukathida Ubol, Departmentof Microbiology, Faculty of Science, Mahidol University, 272Rama VI Rd., Ratchatewee, Bangkok 10400, Thailand. Fax:662–644–5411. E-mail: scsul@mahidol.ac.th

Various types of DNA- and RNA-viruses stimulateapoptosis in the target cells. Rabies virus, a member ofan enveloped RNA virus of the family Rhabdoviridae,that causes a dreadful ancient disease, hydrophobia, isone of the known viruses that can readily induce pro-grammed cell death. The induction of apoptosis byRABV is shown to be dependent on the strain of thevirus, the route of inoculation and the type of host cell(30, 33, 35). RABV-induced apoptosis is mainly foundin a particular experimental condition. This death ismediated by glycoprotein and matrix protein (14, 27).During natural RABV infection neuropathological anddegenerative neuronal changes are not prominent (13).Therefore, the role of apoptosis in natural rabies virusinfection is unclear, unlike that of HIV infection inwhich apoptosis facilitates severity of infection (26).Evidence suggests that successful RABV neuro-inva-sion is dependent on neuronal death suppression (17).This is supported by the pathogenicity of RABV vari-ants being inversely correlated with apoptosis (22, 23).Moreover, investigations found that apoptosis may regu-late the neuropathogenesis of rabies by interfering withdissemination of RABV within the infected brain (2,10). Even though apoptosis is not commonly found innatural RABV infection, the severity of an intracerebralinfection of neonatal mouse model correlates to thedegree of neuronal apoptosis (33). Therefore, no matterwhether apoptosis plays a role as a defensive ordestructive mechanism in RABV infection a greaterunderstanding of apoptosis in an experimental modelmay partly provide insights into the pathogenesis ofrabies.

In the present study, we have used cDNA arrayanalysis to screen for apoptotic and apoptotic-relatedgenes which were activated in suckling mouse brainpost-intracerebral injection with a street strain ofRABV. We were able to demonstrate upregulation ofvarious types of pro-apoptotic genes which are mem-bers of the commitment and execution phase.

Materials and Methods

Viruses. The primary isolate of the rabies virus usedin this study was obtained from the Queen SaovabhaMemorial Institute. One gram of the infected brainfrom a rabid dog was homogenized in 2 ml of PBS,clarified by centrifugation and filtered through 0.2 µmmembrane to eliminate the contaminated bacteria orother microorganisms. The filtrate was kept at �80 Cas stock virus.

Virus infection of mice. One-day-old Swiss albinomice were intracerebrally inoculated with approximately10 µl of 106/ml of the primary isolate of RABV. On

day 2, 4 and 6 after infection, mice were euthanized andtheir brains were removed, kept at �80 C and subjectedto RNA extraction on the next day.

Poly A� RNA isolation and elimination of contami-nated DNA. Total cellular RNA was isolated from themouse brain using a Nucleospin column (Clontech)according to the manufacturer’s instructions. The resid-ual DNA contamination was removed by treatment ofthe RNA with RNase-free DNase supplied by the manu-facturer. Fifty micrograms of the total RNA was sub-jected to poly A� RNA enrichment using Oligo-dTbeads (Clontech). The poly A� RNA was immediatelyused for 32P-labeled single-strand cDNA synthesisaccording to the manufacturer’s instructions.

cDNA array hybridization. The highly labeled sin-gle-strand cDNA, synthesized from the mock-infectedand the RABV-infected mouse brain poly A� RNA, wasdirectly hybridized with the Atlas mouse 1.2 Array andAtlas mouse 1.2 Array II (Clontech) according to themanufacturer’s recommendations. The hybridizedmembranes were autoradiographed. The density of thehybridization spot was analyzed using the Atlas Image2.7 (Clontech) and normalized to the expression of thehousekeeping gene glyceraldehyde 3-phosphate dehy-drogenase (G3PDH). An alteration of gene expressionwas confirmed by RT-PCR.

Reverse transcription and polymerase chain reac-tion. Total RNA was purified from mouse brain har-vested at various times after inoculation using Trizoland phenol/chloroform extraction. The precipitatedRNA was dissolved in DEPC-treated water and wasthen subjected to the first strand cDNA synthesis beforebeing further amplified by PCR using specific primers(40). RABV-N gene primers were sense 5' CAC CTCTAC AAT GGA TGC CG 3' and antisense 5' GCTCAA CCT ATA CAG ACT CA 3'. Fas-L receptorprimers were sense 5' GAG AAT TGC TGA AGA CATGAC AAT CC 3' and antisense 5' ATG GCT GGAACT GAG GTA GTT TTC AC 3'. Caspase-1 primerswere sense 5' TAT GGA CAA GGC ACG GGA CCTATG 3' and antisense 5' CCA GCA GCA ACT TCATTT CTC TG 3'. Calpain 2 primers were sense 5' GCTTGG CTG CTC TAT CGA TAT CAC C 3' and anti-sense 5' CTG GGT CAA CCG TGT TCC AGC TG 3'.The PCR products were confirmed by hybridizationwith specific labeled probes. The specific probes for Ngene, caspase-1 and calpain 2 were 5' CTT GAT CCTGAC GAT GTA TG 3', 5' CTG TGT TGC AGA TAATGA GGG CAA GAC G 3' and 5' GAG CCG AGGTTG AAA GTT CAG G 3' respectively. The level ofgene expression was estimated by a densitometer andexpressed as a ratio relative to β-actin gene, an internalcontrol, expression.

424 S. UBOL ET AL

Detection of apoptosis in the mouse brain by theTUNEL assay. The harvested infected or mock-infectedbrains were fixed in a formalin buffer and embedded inparaffin. The embedded brains were sectioned and sub-jected to TUNEL staining as described (33). Briefly,sections were deparaffinized, rehydrated, permeabilizedand stained with the TUNEL reaction mixture. The flu-orescein-labeled dUTP was transferred to the openedend of the fragmented DNA by the activity of the termi-nal deoxynucleotidyl transferase (TdT). The fluores-cein-stained signal was further amplified by stainingwith an anti-fluorescein-conjugated alkaline phos-phatase. The positive signals were developed by addinga substrate containing 4-nitroblue tetrazolium chlorideand 5-bromo-3-indolyl-phosphate. The apoptoticnuclei were identified by light microscopy.

Results

Reproducibility of the cDNA Array HybridizationTo determine the reproducibility of hybridization,

two independent hybridizations were assessed by divid-ing one RNA sample into two batches that were tran-scribed into 32P-labeled cDNA and then hybridized withtwo membranes. As shown in Fig. 1, the overall corre-lation between the two data sets was very high

(r2�0.81, P�0.05). These data demonstrate a highlevel of reproducibility for the technique.

Screening of Apoptotic and Apoptotic-Related GenesUpregulated in Infected Neonatal Mouse Brain

In order to ensure the induction of apoptosis, neonatalmice were intracerebrally inoculated with RABV. Theappearance of fragmented nuclei was detected using theTUNEL assay. As illustrated in Fig. 2, massive apo-ptotic nuclei were shown on day 6. However, TUNEL-positiveness was detected in some of the infected miceon day 2 and was clearly found in every infected mouseon day 4 (data not shown). This suggests that apoptosiswas initiated at around day 2. The infected neonatesshowed neurological symptoms such as inability tomove forward properly and inability to swallow, whichwas detected by the rapid reduction of milk in theirstomachs.

To screen for genes that may participate in RABV-induced death of the central nervous system, the level ofapoptotic and apoptotic-related gene expression wasscreened and semi-quantitated using cDNA array analy-sis. This experiment was performed using a pool ofRNA extracted from two neonatal mouse brains at eachtime point. To identify the apoptotic genes whichshowed large differences in expression between the

425POTENTIAL APOPTOTIC GENES INVOLVED IN RABIES VIRUS INFECTION

Fig. 1. Scatter plot analysis of the reproducibility of the cDNA array analysis.Two Atlas mouse 1.2 arrays were hybridized with 32P-labeled cDNA generatedfrom the same RNA sample. The solid line represents the predicted “line ofidentity.” The dashed line shows 2.5-fold difference between the two arrays.

426 S. UBOL ET AL

Fig. 2. Detection of apoptotic nuclei in RV-infected suckling mouse brains by TUNEL staining. A: Mock-infectedbrain, B: brain tissue from day 6 post-infection. Arrows indicate apoptotic nuclei.

Table 1. Expression level of apoptotic and apoptotic-related genes on day 6 post-infection

Gene Gene Bank Level of expression

Voltage-gated potassium channel U70068 5.00�0.28Neuronal type calcium channel AF042317 5.50�1.05

TNF-receptor, member 1 Ll26349 15.95�4.60TNF-receptor, member 5 M83312 6.80�2.26Fas-ligand receptor M83649 11.60�5.18

BAD L37296 10.35�4.60BAK Y13231 4.61�2.26Programmed cell death protein 6 U49112 3.06�0.49Caspase-1 L28095 4.15�0.92Caspase-3 U19522 4.15�0.64

Annexin V D63423 4.96�2.18Annexin XI U65986 3.75�0.35Synexin L13129 5.18�1.45Cytochrome c AF037371 15.10�5.65Clusterin L08235 3.00�1.27

Calpain 2 D38117 7.10�0.42Cathepsin D X53337 6.80�0.85Cathepsin L X06086 2.55�0.49

Cyclin-dependent kinase 5 S82819 6.60�1.98ERK M61177 3.65�1.63P38MAPK U10871 3.9�0.4

infected mouse and the mock-infected controls, geneswith a mean level of expression that was 2.5-fold higheror lower in the infected group compared to the controlwere selected. Various types of potential apoptoticgenes activated in the neonatal mouse brains inresponse to RABV infection are shown in Table 1.These genes were death receptors (Fas-L receptor,TNF-receptor), bcl-2 family, caspases, lysosomal pro-teases (cathepsin D, L), annexins, ion-channel, calpainand signaling molecules. The level of expression ofthese genes ranged from 2.5–15-fold difference com-pared to control.

In order to study the sequence of the apoptoticevents, the time course of expression of these genes wasmonitored on day 2, 4 and 6 post-infection. As demon-strated in Table 2, Fas-L receptor, BAD, caspase-1, cal-pain 2, ERK and p38MAPK were activated as soon as 2days after infection. By day 4, 18 apoptotic genes,

functioning in both the commitment and the executionphase, were significantly activated. A total of 21 geneswere upregulated by day 6. According to this data, themajority of apoptotic genes were stimulated betweenday 2 and 4. Most of the genes stimulated on day 2were members of the immune-related apoptotic media-tors such as caspase-1 and Fas-L receptor.

However, not only were the apoptotic genes stimulat-ed but also the anti-apoptotic genes. Lipocortin I, sen-trin and Akt-kinase were significantly upregulated byday 6 (Table 3). Lipocortin I and sentrin are inhibitorsof TNF and Fas-mediated apoptosis whereas Akt-kinase is a survival signal which inhibits Bid activation.

Confirmation of Gene Expression Alteration by RT-PCRTo validate the alteration of apoptotic gene expression

detected by array analysis, RT-PCR of genes upregulat-ed on day 2 post-infection was performed. The analysis

427POTENTIAL APOPTOTIC GENES INVOLVED IN RABIES VIRUS INFECTION

Table 2. Upregulation of apoptotic and apoptosis-related genes at differenttime points after infection

Gene Day 2 Day 4 Day 6

Voltage-gated potassium channel � � �Neuronal type calcium channel � � �

TNF-receptor, member 1 � � �TNF-receptor, member 5 � � �Fas-ligand receptor � � �

BAD � � �BAK � � �Programmed cell death protein 6 � � �Caspase-1 � � �Caspase-3 � � �

Annexin V � � �Annexin XI � � �Synexin � � �Cytochrome c � � �Clusterin � � �

Calpain 2 � � �Cathepsin D � � �Cathepsin L � � �

Cyclin-dependent kinase 5 � � �ERK � � �P38MAPK � � �

��The expression level was upregulated significantly. ��There was noalteration of gene expression.

Table 3. List of apoptosis inhibitor genes that were upregulated on day 6 post-infection

Level of expression Gene

(means�SD)Function Reference

Lipocortin I 3.46�0.78 Inhibit TNF-mediated apoptosis 42Sentrin 5.08�1.94 Inhibit Fas-mediated apoptosis 25Akt-kinase 2.61�0.83 Survival signal 15, 20, 46

was done on the total RNA prepared from individualmice. As shown in Fig. 3A, the replication of RABV inthe suckling mouse brain was demonstrated using the Ngene expression level. The low level of the RABVtranscription began on day 1 and became significantlyelevated on day 2 post-infection. Significant elevationsof FAS-L receptor, caspase-1 and calpain 2 RNA syn-theses were observed on day 2, which confirmed data

from cDNA analysis (Fig. 3, B–D). Activation of theseapoptotic genes correlated with the level of the RABVreplication.

Discussion

In this study, the gene expression profile of RABV-infected suckling mouse brain was compared to the cor-

428 S. UBOL ET AL

Fig. 3. Upregulations of Fas-L receptor, caspase-1 and calpain 2 genes were confirmed by RT-PCR. A: Determination ofrabies virus replication by N-gene RT-PCR, B: detection of Fas-L receptor gene expression, C: semiquantitation of cas-pase-1 gene expression, D: expression level of calpain 2 gene.

responding control, using cDNA array analysis. At theinitial phase of data analysis, only apoptotic and apo-ptotic-related genes were reported since massive apo-ptotic nuclei can be clearly seen in our experimentalmodel. These apoptotic cells may indeed be infectedneurons as well as blood cells since unperfused-infectedbrains were used. We found that various potentialgenes involved in the commitment phase and execution-ers were activated. Genes of the commitment phasewere lysosomal proteases (cathepsin D, L), death recep-tors (Fas-L receptor, TNF-receptor), Ca2�-dependentneutral proteases (calpain), bcl-2 family members(BAD, BAK) and signaling molecules (ERK,p38MAPK). Cytochrome c and caspase-3 were repre-sentatives of executioners. In addition, the inflammatorycaspase, caspase-1, was also significantly upregulatedduring the early phase of apoptosis. Besides severalapoptotic modulators, the expression of certain anti-apoptotic genes, including lipocortin, sentrin and Akt-kinase, were demonstrated to be elevated during the latephase of apoptosis.

Apoptosis promoted by pro-inflammatory cytokinesis mediated via an inflammatory caspase, caspase-1.This caspase functions as an activator of IL-1β, which inturn indirectly stimulates caspase-3 through IL-6 upreg-ulation (43, 45). Expression of the IL-1β gene was sig-nificantly upregulated (3.6-fold, data not shown) inRABV-infected mouse brains. Pro-inflammatorycytokines-facilitated apoptosis has been shown in bothHIV-1-associated dementia and Parkinson’s disease,respectively (24, 32). Moreover, upregulation of deathreceptor, TNF-receptor and Fas-L receptor, and theiranti-apoptotic genes, lipocortin and sentrin, suggestedthe role of these receptors on RABV-induced apopto-sis. Ligand-receptor mediated apoptosis, a TRAIL-dependent pathway, in Lyssavirus infection has beenreported and found to involve caspase-8 activation (14).Whether these immunological molecules participated ininflammation or mediated apoptosis in our modelrequired further investigation.

In addition to caspases, apoptosis may be signaledby other proteases such as cathepsins and calpains.Calpain is a ubiquitous cysteine protease with twomajor isoforms, m- and µ-calpain. The m-calpain isexpressed in interneuron while µ-calpain is found in theprincipal neuron. During RABV infection, the expres-sion of m-calpain, calpain 2, was increased significantly.Calpains are activated by calcium influx and facilitatedby calpastatin degradation by caspases (3, 29). The m-calpain is shown to modulate cell death by direct Bidcleavage, by activation of calcineurin A and by stimula-tion of killer kinase (16, 21, 41). The upregulation ofm-calpain implied the role of this molecule in apoptosis

during RABV infection.Several lysosomal proteases of the cathepsin family

are actively involved in apoptosis. Cathepsin L hasbeen shown to activate Bid and caspase-3 during apo-ptosis in HeLa cells and cultured cortical neuronsrespectively (5, 7). Similarly, cathepsin D also activatescaspases (caspase-9 and -3) via Bid cleavage (12). Fur-thermore, cathepsin D has been shown to trigger Bax,which subsequently leads to release of apoptosis-induc-ing factor, AIF (4). During RABV infection, Bad, Bakand cytochrome c gene expression were increased sig-nificantly, suggesting that these molecules may stimulatedownstream caspases, caspase-3 in our experimentalmodel. Even though information on the upregulationof Bid was not available due to the absence of Bid geneon the array used, an increased expression of Bidinhibitor, Akt-kinase, and the upregulation of Bid acti-vators, calpain, cathepsin and caspase-1, suggestedinvolvement of Bid during RABV-induced apoptosis.The activated Bid plays a critical role in inducing theoligomerization of Bak and/or Bax, which is known tostimulate neuronal death (18). Bax and AIF-relatedapoptosis have been demonstrated in RABV-infectedcultures as well as in an in vivo model (9, 36, 37). Bothcathepsin D and L genes expressions were upregulatedin the CNS during the RABV infection, suggesting thatRABV infection induced lysosomal disruption and acti-vation of lysosomal-mediated apoptosis. These lysoso-mal proteases may then activate a cascade of bcl-2 pro-teins and mitochondrial leakage. Whether this processoccurred during RABV infection required further inves-tigation.

The cascade of the bcl-2 family and caspases isknown to be controlled by several upstream signalingmolecules. These signaling molecules such as ERK-1(MAPK), P38MAPK and cyclin-dependent kinase 5have been shown to activate neuronal death in varioussystems (28, 44). Therefore, the upregulation of thesesignaling molecules during RABV infection may serveas a critical point for infected cells to enter into a life ordeath process.

In addition to the apoptotic genes mentioned above,annexin genes, as well as a voltage-gated potassiumchannel were also upregulated. Various types of apo-ptotic stimuli enhance certain ionic effluxes, includingK� and Cl�. K� efflux is an essential process of cellularshrinkage, as a consequence of the downstream caspaseactivation and DNA fragmentation (31, 39). IncreasedK� channel gene expression implied that K� efflux mayoccur during RABV infection and may mediate cellularshrinkage during RABV-induced apoptosis.

In conclusion, natural rabies virus (RABV) infectionstarts with peripheral spreading of RABV from infected

429POTENTIAL APOPTOTIC GENES INVOLVED IN RABIES VIRUS INFECTION

wound to central nervous system. Our study bypassedperipheral effects and investigated direct interactionbetween RABV and its targets. It may not reflect natu-ral infection but it demonstrated another possible eventof virus and host interaction. Our investigation suggestedthat RABV stimulated more than one mode of death inthe central nervous system. During rabies virus infec-tion, multiple types of cells undergo apoptosis, includingRABV-infected cells, bystander cells and infiltrated Tcells (2). This death is stimulated by two independentsets of factors, immunological factors and proteases.The immunological factors were caspase-1, IL-1β, Fas-L receptor and TNF-receptor. The others may occurthrough lysosomal proteases and Ca2�-dependent neu-tral protease activities. Like apoptosis induced by otherstimuli, RABV-induced death involves mitochondriaand caspases. However, it can not be ruled out that agene which falls in the apoptosis category found in ourscreening may also function in several other processes.To clarify this issue, it is necessary to investigate the insitu expression of the pro-apoptotic category in apoptot-ic brain cells.

We thank Prof. Svasdi, J. and Assoc. Prof. Sophasan, S. forediting the manuscript. This work is supported by ThailandNational Center for Genetic Engineering and Biotechnologygrant number BT-B-06-MM-14-4505.

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431POTENTIAL APOPTOTIC GENES INVOLVED IN RABIES VIRUS INFECTION