Programmed Neuronal Cell Death Induced by HIV-1 Tatand MethamphetamineLI QI,1,2y LU GANG,3y KWONGWING HANG,3 CHOI HEUNG LING,3 ZENG XIAOFENG,2,3 LI ZHEN,2*YEW DAVID WAI,3 AND POON WAI SANG3

1Department of Psychiatry, University of Hong Kong, 21 Sasson Roads, Pokfulam, Hong Kong Special Administrative Region, China2School of Forensic Medicine, Kunming Medical University, Kunming, Yunnan Province, China3Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region, China

KEY WORDS HIV-1 tat; methamphetamine; SH-SY5Y; apoptosis; autophagy; programmed celldeath

ABSTRACT Apoptosis and autophagy are the two major types of programmed cell death (PCD)in neurons. Homeostatic autophagy often precedes apoptosis, and when apoptosis is blocked, thefailure to keep homeostasis will lead to necrosis instead. It has been reported that human immuno-deficiency virus (HIV) infected methamphetamine (Meth) abusers represent greater neuropa-thological abnormalities than Meth abusers or HIV-positive non-Meth users. Recent publicationssuggest that Tat and Meth when administered together result in greater neuronal damage thanwhen administered separately. However, the cellular events of the combined Tat-Meth effect havenot yet been fully characterized. Therefore, we investigated the effects of Tat and/or Meth on apo-ptosis and autophagy to elucidate whether PCD was involved in Tat and/or Meth-induced neuronaldamage. Annexin-V-FITC/PI staining assay was used to detect cellular apoptosis using a neuro-blastoma cell line SH-SY5Y. Cellular ultrastructural changes were observed under transmissionelectron microscopy (TEM). Flow-cytometric data showed apoptosis following Meth treatment, andmore extensive apoptosis with Tat 1 Meth treatment. The most important finding was that theautophagosome and/or multilamellar bodies (MLBs) were most pronounced with Tat 1 Meth treat-ment, were less so with Meth treatment, and infrequent with Tat treatment. This suggeststhe involvement of autophagy and apoptosis in Tat with Meth-elicited cell damage. However, therelation between apoptosis and autophagy remains unknown in this experiment. Further researchis needed to analyze the relation among related molecules. A thorough understanding of this multi-faceted relationship will be critical for the assessment of therapeutic modalities for patients withHIV with drug abuse.Microsc. Res. Tech. 74:1139–1144, 2011. VVC 2011 Wiley Periodicals, Inc.

INTRODUCTION

Methamphetamine (Meth) is a highly abused psy-chostimulant and its use is a known risk factor for con-tracting human immunodeficiency virus-1 (HIV-1)infection. HIV-positive individuals with Meth abuse of-ten have earlier and more severe symptoms than thosewho do not use Meth (Reiner et al., 2009). It is thoughtthat the combination of Meth abuse and HIV infectionmay lead to substantial alterations in brain functions.Among the HIV-1 proteins, only the Tat (transactivatorof transcription) protein is crucial to HIV-1 for its repli-cation (King et al., 2010); thus, the potential interac-tion effect of Tat and Meth has become an emergingproblem in HIV pathogenesis.

The mechanisms responsible for HIV-induced neurondamage have not been well defined. However, HIV-Tatis toxic when released from infected lymphoid and glialcells. Tat-infected glial cells produce a variety of neuro-toxins, including proinflammatory cytokines, particu-larly tumor necrosis factor alpha (TNF-a) and interlu-kine-1 beta (IL-1b), which are detrimental to neurons(Lafrenie et al., 1997). Tat can also induce astrocytes torelease inducible nitric oxide synthase (iNOS), whichmay diffuse from astrocytes to neurons and initiateapoptotic events in neurons (Blond et al., 2000; Hori

et al., 1999). Recent findings demonstrate that theinteraction of Tat with neuronal surface receptors isdependent onN-methyl-D-aspartate receptor (NMDAR)activity, regulated by NMDAR subunit 2A (NR2A)(King et al., 2010). This may initiate apoptosis in neu-rons and contribute to the pathogenesis of neurocogni-tive impairment in HIV-infected individuals.

Meth is a psychostimulant with high abuse potential.Abusers show significant increases in levels of activatedmicroglia in the midbrain, striatum, thalamus, and cor-tex (Sekine et al., 2008). Meth may also be toxic to thedopaminergic reward pathway. Evidence shows thatMeth has dopaminergic activation properties, eitherthrough inhibiting the reuptake of dopamine (DA) or bydisrupting the vesicular storage of DA (Goodwin et al.,2009; Guillot et al., 2008; Westphalen and Stadlin,

yL.Q. and L.G. contributed equally to this work.

*Correspondence to: Li Zhen, School of Forensic Medicine, Kunming MedicalUniversity, 191 West Renmin Road Kunming, Yunnan Province, China.E-mail: lizhenlaura@126.com

Received 27 January 2011; accepted in revised form 10 February 2011

Contract grant sponsor: National Natural Science Foundation of China(NSFC); Contract grant number: 30660202; Contract grant sponsor: Yunnan Pro-vincial Department of Science & Technology; Contract grant number: 2006GH22

DOI 10.1002/jemt.21006

Published online 11May 2011 inWiley Online Library (wileyonlinelibrary.com).

VVC 2011 WILEY PERIODICALS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 74:1139–1144 (2011)

2000). The chronic increase in dopamine leads to over-stimulation of the postsynaptic neurons, which cancause apoptosis, autophagy, or cell death in these neu-rons (Deng et al., 2001). It is known that apoptosis andautophagy are the two major types of programmed celldeath (PCD) in neurons (Schweichel and Merker, 1973).Homeostatic autophgy often precedes apoptosis, andwhen apoptosis is blocked, the failure to keep homeosta-sis will lead to necrosis instead (Xie et al., 2010).

Although many studies have focused on the individ-ual effects of Meth and HIV-tat on the brain, fewexperiments have investigated their interaction. Thereis much evidence showing that Tat can damage the pro-jections of the striatum, including nigrostriatal dopa-mine (DA) neurons (Zhu et al., 2009). It is well knownthat the striatum is a predominant target of Meth inthe brain (Jayanthi et al., 2009). Experimental evi-dence suggests that Tat can cause toxicity to dopamineneurons and this toxicity may be synergistic with Meth(Cai and Cadet, 2008). However, the mechanismsunderlying the synergistic effects of Meth and Tat onneurons remain unclear. Recent studies show that Tatand Meth act through the glial cells (astrocytes ormicroglia, or both) to produce such a synergistic effect(Theodore et al., 2006). However, it is not clear whetherTat and Meth have a direct synergistic effect on dopa-mine neurons. In vivo studies suggest that Meth abus-ers infected with HIV may be at increased risk for ba-sal ganglia dysfunction. In addition, the cellular eventsof the combined Tat-Meth effect have not been morpho-logically characterized. Therefore, we investigated theeffects of Tat- (50 ng/mL) treated, Meth- (500 lM)treated, and TAT1Meth-treated cells on apoptosis and/or autophagy to elucidate whether PCD was involvedin Tat and/or Meth-induced neuronal damage.Annexin-V-FITC/PI double staining assay was used todetect cellular apoptosis using a neuroblastoma cellline SH-SY5Y, which is known to display the morpho-logical, biochemical, and electrophysiological proper-ties of dopaminergic neurons. Cellular ultrastructuralchanges were observed under TEM. A thorough under-standing of the mechanisms that lead to this synergis-tic effect on dopaminergic-like neurons could also leadto the development of therapeutic modalities forpatients with HIV with drug abuse.

MATERIALS AND METHODSCell Culture

Neuroblastoma SH-SY5Y cells, in 8–16 passages,were grown in a 1:1 mixture of DMEM and Ham’s F12medium supplemented with 10% fetal bovine serum,and 100 g/mL of streptomycin. Monolayer cultureswere incubated in 75-cm2 tissue culture flasks at a den-sity of 104 cells/cm2, in a 95% air and 5% CO2 humidi-fied atmosphere at 378C. The medium was replacedthree times a week.

Tat and Meth Treatments

The cells were incubated with medium only (controlgroup), Tat (50 ng/mL), Meth (500 lM), and Tat1Meth.The chosen concentrations of Tat and Meth were athreshold concentration that would only cause mar-ginal toxicity to neurons when given alone (Theodoreet al., 2006; Turchan et al., 2001). After 24 h at 378C,

all the treatments were stopped and the cells wereappropriately post-treated according to the differentexperiments.

Flow Cytometric Analysis UsingAnnexin Vand PI

SH-SY5Y cells were centrifuged to remove the me-dium, washed with PBS, and stained with annexin V-FITC and propidium iodide (PI) in a binding buffer (10mM Hepes, 140 mM NaCl, 2.5 mM CaCl2). Ten thou-sand events were collected for each sample. Stainedcells were analyzed using a flow cytometer (CoulterEpics AltraTM, Beckman Coulter, USA). Green andred fluorescence were analyzed on the FL1 (525 nmBP) and FL2 (575 nm BP) channels, respectively. Thepercentage of red and green fluorescence was quanti-fied using Expo32TM software (Beckman CoulterTM)and annexin V and PI were expressed as the green/redfluorescence ratio. The experiment was performed intriplicate and three independent experiments wereconducted. The results were expressed as the meanpercentage of apoptosis cells. The results were ana-lyzed using ANOVA with four (groups) 3 two (annexinV and PI) factors, followed by posthoc Bonferroni test.The criterion for statistical significance was set as P <0.05. Analyses were conducted using the statisticalsoftware SPSS for Windows (version 16.0).

TEM

After treatment of cells with Tat and/or Meth for 24h, the transwells were washed twice with 0.1M phos-phate buffer (PB) at pH7.4 before fixing the cells with2.5% glutaraldehyde for 2 h at 48C by immersing thewhole transwell into the fixation. They were then post-fixed with 1% osmium tetroxide for 30 min, and washedwith 0.1M phosphate buffer (pH 7.4), dehydrated witha series of graded alcohol (20–100%), embedded inSpurr’s embedding medium, and then sectioned on aReichert-Jung ultramicrotome. Thin sections werestained with uranyl acetate and lead citrate and exam-ined with a Hitachi 7100 transmission electron micro-scope operating at 50 kV.

RESULTSFlow Cytometric Analysis Using

Annexin Vand PI

The results indicated that only annexin V1/PI-showed a significant difference between the fourgroups (F 5 31.37, P < 0.0001) (see Fig. 1). Annexin Vpositive and PI negative suggested that cells were inearly apoptosis. When cells are in late apoptosis or al-ready dead, both annexin V and PI are positive. How-ever, there was no obvious cell death (annexin V1/PI1) in any of the groups (see Fig. 1).

Treatment with Tat alone resulted in 25.7% 6 0.0024cell apoptosis, which was not significantly differentfrom that in controls (23.8% 6 0.0069) (see Fig. 2). Cul-tures treated with 500 lM Meth had a significantlyhigher degree of apoptosis (28.4% 6 0.0089) from thatin controls. As expected, there was a profound enhance-ment of apoptosis in cultures exposed to both Tat andMeth (32.8%6 0.0072) (see Fig. 2). According to posthoctests, cells with Meth1Tat treatment showed signifi-cant apoptosis when compared with the control, Meth,and Tat treatments (P < 0.01) (see Fig. 2).

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TEM

In control cells and Tat-treated cells, a prominentnucleolus was observed which was surrounded by asmall border of electron-dense, well-preserved cyto-plasm, mitochondria, some rough endoplasmatic reticu-lum (RER), and free ribosomes (Fig. 3A). TEM was per-formed to detect the formation of autophagosomesresulting from a small portion of the cytoplasm engulfedby an isolation membrane. In this experiment, double-membranes and giant autophagosomes filled withdegraded organelles, were frequently observed in Meth-treated cells (Figs. 3B and 3D). In addition, AU andmultilamellar bodies (MLBs) were found in Tat 1Meth-treated cells (Figs. 3C and 3E). MLBs are definedas cytoplasmic organelles containing at least threedistinct circumferential concentric membrane lamellae.The presence of MLBs indicates a form of mature,single-membrane autophagosome.

DISCUSSION

The key finding is that a combination of Meth andTat caused a significant increase in apoptosis in human

neuroblastoma cells, which was clearly evident afteronly 24 h of exposure. In addition, synergistic effectswere found on micro-morphological alterations, such asautophagy and MLB, in cells exposed to both Meth andTat.

An in vivo experiment demonstrated that Meth syn-ergizes with HIV-Tat. Rats receiving HIV-Tat followedby Meth have shown an almost fivefold reduction indopamine levels compared with animals treated witheither Meth or HIV-Tat alone (Cass et al., 2003). How-ever, it is not yet known whether the enhancement ofMeth and HIV-Tat toxicity is due to a loss of dopamineneurons in the substantial nigra or to the degenerationof striatal dopaminergic afferents. At present, it isknown that exposure to both Meth and HIV-Tat has aneffect on tyrosine hydrocyclase activity, which is a rate-limiting enzyme in the synthesis of dopamine (Zauliet al., 2000). The mechanism responsible for this syner-gistic toxicity by Meth and HIV-Tat has not yet beenelucidated. In vitro studies using human neuronalculture have shown that Meth1Tat- induced toxicityimpairs mitochondrial function (Maragos et al., 2002).Mitochondrial dysfunction has been implicated in

Fig. 1. Flow cytometric analysis of apoptotic cells using annexinV-FITC & PI. SH-SY5Y cells were incubated with annexin V-FITC ina buffer containing PI and analyzed by flow cytometry. The picturesfrom top left to bottom right show the control (untreated SH-SY5Ycells), Meth treatment, Tat treatment, and Tat 1 Meth treatment.

The lower left quadrant of each picture contains the vital cells, thelower right quadrant contains the apoptotic population (annexin V1/PI2), and the upper right quadrant contains the late apoptosis ordead cells (annexin V1/PI1). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

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many neurodegenerative disorders (Beal, 2000; Orthand Schapira, 2001). Recent studies have shown thatmitochondria-formed oxidants are mediators for molec-ular signaling in the apoptotic pathway (Niizumaet al., 2009).

In the central nervous system (CNS), PCD, includingapoptosis, autophagy, and cytoplasmic cell death, isan important process during the development of thenervous system (Hurle et al., 1996). Apoptosis is welldocumented and has been recognized to be an essentialprocess during the development of neurons in the CNS(Strange et al., 2001). Chromatin condensation andsegregation into sharply delineated masses on thenuclear envelope are typically observed at the onset ofapoptosis (Sun et al., 1994). Autophagy is an evolutio-narily conserved process that plays a prosurvival rolein cell death in pathophysiological conditions. How-ever, extensive or inappropriate activation of autoph-agy results in autophagic cell death. Autophagy refersto a type of cell death in which the cytoplasm is activelydestroyed by lysosomal enzymes before nuclearchanges become visible. The most characteristic fea-tures of autophagy are the appearance of large auto-phagic vacuoles of lysosomal origin in the cytoplasm(Gonzalez-Polo et al., 2005). However, there is no clear-cut distinction between apoptosis and autophagy.Recent studies have focused on the cross-talk betweenapoptosis and autophagy and have suggested that theytend to inhibit each other (Kroemer et al., 2010; Marinoet al., 2010). When autophagy is blocked, apoptosisensues, and when apoptosis is blocked, autophagy isactivated as a cell-survival mechanism. However, whenall the subcellular organelles are self-eaten, the celleventually dies.

In the current experiment, we used annexin V as anearly cell apoptosis marker and propidium iodide (PI)as a cell death marker to measure the coeffects of Methand HIV-Tat on SH-SY5Y. Annexin V is a Ca21 depend-

ent phospholipid-binding protein that can bind to cellswith exposed phosphatidylserine (PS), which is trans-located to the outer leaflet of the membrane duringearly apoptosis. The chosen concentrations of Tat andMeth were determined to be the thresholds andresulted in toxicity only slightly greater or equal tothat in vehicle-treated cells. In this experiment, withTat and Meth treatment or Meth treatment only,significant apoptosis was induced in SH-SY5Y cells.However, we found that with Tat treatment only, therewas no obvious apoptosis when compared with the cellstreated with medium only. Our study employed athreshold dose of Meth and Tat, which individuallywould not cause an obvious apoptosis. Therefore, theobserved discrepancies with the Meth treatment musthave been due to the different cell types and the doseemployed. However, with the combined treatment ofTat with Meth, an obvious apoptosis was found. Thissuggests that there is a synergistic toxicity with Methand HIV-Tat.

However, only giant autophagosomes (AU) filled withdegraded organelles were frequently observed in theMeth-treated cells. In addition, AU and multilamellarbodies (MLBs), a mature form of autophagosomes, werefound in Tat1Meth-treated cells. Although we foundenhanced apoptosis in Tat1Meth-treated cells, we didnot find any apoptotic bodies in these cells. Previousstudies have shown that depolarized mitochondria aremore likely to be eliminated by autophagy (Wang andKlionsky, 2011). Therefore, in this experiment, reduc-tions in mitochondrial membrane potential could be anautophagy trigger in either apoptotic or necrotic cells.Recent studies have suggested that autophagy has adual role, either facilitating or inhibiting apoptosis, indrug-induced apoptosis (Djavaheri-Mergny et al., 2010).Autophagy and apoptosis do coexist in MA1Tat-treatedcells, which might imply that neurons either try toescape apoptosis or die by autophagy. It would be inter-esting, therefore, to explore the interaction activitybetween autophagy and apoptosis in future studies.

Extensive literature in recent years has developedour knowledge of the cellular and molecular mecha-nisms of apoptosis in neurons. Recent reports suggestthat autophagy and apoptosis can be induced by thesame stimuli and they may share similar effectorsand regulators, and thus a complex cross-talk existsbetween these two processes (Eisenberg-Lerner et al.,2009). Mitochondrial reactive oxygen species (ROS)play an important role in the regulation of autophagyand apoptosis. Excessive ROS production represents adeath threat for the cell and is essential for autophagyinduction. Furthermore, the effect of ROS is partiallyPI3K dependent, and PI3K plays an important role inthe prevention of apoptosis (Franke et al., 1997, 2003;Mammucari and Rizzuto, 2010). Autophagy can lead tothe execution of apoptotic or necrotic cell death, pre-sumably through a common regulator such as proteinsfrom the Bcl-2 family (Oberstein et al., 2007). Thereare multiple and complicated connections between theapoptotic and autophagic processes, and the two phe-nomena jointly decide the fate of the cell. Furthermore,the cross-talk between apoptosis and autophagy is akey factor in the outcome of death-related pathologiessuch as HIV. However, current knowledge of the molec-ular intersections between autophagic and apoptotic

Fig. 2. Cell apoptosis (annexin V) was quantified in the SH-SY5Ycell line that was exposed for 24 h to either 500 lM Meth or 50 ng/mLTat alone or in combination. Although treatment with Tat caused nosignificant apoptosis, exposure to Meth induced a slight increase incell apoptosis compared with controls. However, when cells wereexposed to both Meth 1 Tat, there was a synergistic enhancement ofapoptosis. *Treatment groups versus control group. ##Meth1Tattreatment group versus other groups (control, Meth, and Tat groupsare included).

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pathways is incomplete. Further research is needed toanalyze the relation between related molecules and, inparticular, to explore the interactional effects of apo-ptosis and autophagy on dopaminergic neurons. Thecomplex interplay between apoptosis and autophagy isnot only of theoretical significance to the researchersbut will impact on the course of treatment for diseasethat is associated with cell death. Hence, a thoroughunderstanding of this multifaceted relationship couldlead to new concepts and novel strategies for interven-tion in HIV with drug abuse.

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