SIMONA OZDEN,§ CATHERINE VIDAL,�� PIERRE CHARNEAU,** AND SYLVIE GRANON††
Unite Postulante “Mycologie Moleculaire,” Institut Pasteur, Paris, France; *Unite Postulante“Macrophages et Developpement de l’Immunite,” Institut Pasteur, Paris, France, †Unite “Retrovirus& Transfert genetique,” Institut Pasteur, Paris, France; ‡Service d’Anatomie et de CytologiePathologiques, Hopital Lariboisiere, Paris, France; §Unite “Epidemiologie & Physiopathologie desVirus Oncogenes,” Institut Pasteur, Paris, France; ��CEA, Service de Neurologie, CRSSA, EPHE,Fontenay-aux-Roses, France; **Groupe “Virologie Moleculaire & Vectorologie,” Institut Pasteur, Paris,France; and ††Unite “Recepteurs & Cognition,” Institut Pasteur, Paris, France
ABSTRACT Complex mechanisms of human immu-nodeficiency virus type-1 (HIV-1) brain pathogenesissuggest the contribution of individual HIV-1 gene prod-ucts. Among them, the Nef protein has been reportedto harbor a major determinant of pathogenicity inAIDS-like disease. The goal of the present study was todetermine whether Nef protein expressed in vivo byprimary macrophages could induce a brain toxicity alsoaffecting the behavior of the rat. To achieve this goalwe grafted Nef-transduced macrophages into the rathippocampus. Two months post-transplantation, weobserved that Nef induces monocyte/macrophage re-cruitment, expression of TNF-�, and astrogliosis. Noapoptotic event was detected. We further demonstratedthat Nef neurotoxicity is associated with cognitive def-icits.—Mordelet, E., Kissa, K., Cressant, A., Gray, F.,Ozden, S., Vidal, C., Charneau, P., Granon, S. His-topathological and cognitive defects induced by Nef inthe brain. FASEB J. 18, 1851–1861 (2004)
Key Words: neuropathology � behavior � bone marrow-derivedmacrophages
Soon after exposure, the human immunodeficiencyvirus type 1 (HIV-1) invades the central nervous system(CNS) and induces AIDS cognitive disorders (1–6).The neuropathological damages commonly known asAIDS cognitive disorders (ADC) are characterized byprogressive deterioration of mental and motor func-tions. When observed, brain lesions mainly affect thewhite matter, the basal ganglia, the cerebral cortex, andthe hippocampus (1, 7–9). However, a non-negligiblepercentage of patients show no lesion but exhibitminor cognitive deficits that may constitute a pathologydifferent from ADC (10). At a cellular level, the mainreservoir for HIV replication are monocyte-derivedmacrophages/microglial cells (11–13). A low level ofinfected astrocytes, neurons, and brain capillary endo-thelial cells has been reported in brain specimens withADC (14–18).
Several lines of recent in vitro and in vivo evidence
suggest that different factors are involved in HIV-1neuropathogenesis. Among them, viral products (Tat,gp120, gp160, Nef), cellular products (acid quinolinic,NO, etc.), cytokine induction (TNF-�, IL-8, INF-�), andexcitotoxic injuries may lead to neurodegeneration orneuronal loss (19–23).
Nef protein is precociously and strongly expressedduring HIV-1 infection (24, 25). Immunohistochemicalstaining has revealed an overexpression of Nef com-pared with other viral proteins in HIV-infected astro-cytes and brain biopsies (26, 27). In SIV-infected mon-keys, it has been shown that Nef expression is essentialto maintain a high replication level of the virus andpromote the development of AIDS (28). In a transgenicmouse model, Skowronski and collaborators (29)showed that the expression of Nef in lymphocytesTCD4� induced modifications in the signaling path-way of those cells leading to their progressive death.Moreover, among the transgenic mice expressing thecomplete coding sequences of HIV-1 in T CD4� cells,only the Nef transgenic mice developed a severe AIDS-like disease, detected in all organs, including the brain(30). Altogether, these data suggest a potentially impor-tant and specific role for Nef in cellular dysfunctionsand its contribution to the development of the neuro-pathology associated with AIDS (31–34).
A highly significant increase in the number of mac-rophages in patients who suffered from ADC has beenreported in postmortem studies, pointing to a possiblerole of macrophage infiltrates for the exacerbation ofneuronal dysfunction (35). Several clinical and exper-imental studies have focused on the production ofchemotactic factors during HIV infection (36–39).Recently, Koedel and collaborators (40) have proposedthat Nef is essential for AIDS neuropathology as amediator for the recruitment of leukocytes. Therefore,
the presence of leukocytes within the brain may serve asa vehicle for the virus and may perpetrate the diseasethrough their production of neurotoxins. Overall, thiscorpus of results led us to investigate the role of the Nefprotein when expressed by some preferential HIVtarget cells (macrophages). In parallel, we performeddirect injection of vector particles known to targetmainly astrocytes in order to see whether the expres-sion of Nef in the astrocytes would be different from itsexpression in macrophages.
The main aim of our research was to determine invivo whether Nef, expressed by macrophages whentransplanted onto the rat brain could induce observ-able early toxicity. To address this, we had efficientlytransduced ex vivo rat primary macrophages with alentiviral vector (41) and showed that, in vivo, trans-plants of transduced macrophages into the rat brainmaintain long-term expression of a control protein(green fluorescent protein, or GFP) for up to 90 dayswithout any significant sign of astrogliosis.
In the present study, we showed a sustained, stable,and noncytotoxic expression of Nef in bone marrow-derived macrophages (BMDM) in culture. This firststep allowed us to graft primary macrophages express-ing Nef onto the rat hippocampus and study theireffects on behavior and histopathology. Our presentresults corroborate the presence of Nef in the hip-pocampus with its histopathological effects includingmonocyte/macrophage recruitment, expression of theproinflammatory cytokine TNF-�, and astrogliosis. Wefound no correlation between the presence of Nef inthe brain and certain apoptotic events. We show thatNef neurotoxicity is associated with cognitive deficitsspecific to hippocampal dysfunction.
MATERIALS AND METHODS
Primary culture of BMDM
Bone marrow cells were collected from femurs and tibias ofadult male Long-Evans rats (Janvier, Le Genest-St.-Isle,France) as described (41, 42). Briefly, cells were flushed outwith a 25 gauge needle into ice-cold phosphate-bufferedsaline (PBS) without Mg2�, Ca2�. The marrow plugs werecentrifuged (1500 g, 10 min, 4°C). After dissociation andelimination of the red cells, bone marrow cells were seededonto 6-well dishes (4�106 cells/mL, Costar, Cambridge, MA,USA) in RPMI 1640 (Seromed, Berlin, Germany) supple-mented with 2 mM l-glutamine, 2 0 mM NaHCO3, 5U/mLpenicillin, 50 �g/mL streptomycin, and 10% heat-inactivatedfetal calf serum. The conditioned medium from L929 (5%) asa source of colony-stimulating factor 1 (CSF-1) was added inRPMI supplemented (43). At day 3, adherent and differenti-ated BMDM were then used for transduction. For immuno-fluorescence studies, glass coverslips were placed into thewells of cultures plates. For in vivo transplantation, the cellswere detached with trypsin-EDTA 1�, washed and resus-pended in serum and CSF-1 free medium at 2.5 � 107
cells/mL.
Lentiviral vector
A three-plasmid expression system was used to generatevector particles by transient transfection of 293T cells using
the calcium phosphate coprecipitation technique as de-scribed (44, 45). We used the vector plasmid encoding anhCMV-driven expression cassette of the GFP or Nef cDNA(TRIP vector). The encapsidation plasmid (p8.2) provides allvector proteins and the VSV-G envelope expression plasmid(phCMV-G) permits the production of vector particles. Thesystem was improved by the deletion of the U3 region of the3� long terminal repeat (LTR) of the DNA used to producethe TRIP vector (46). The vector stocks were treated withDnaseI as described (45). Vector titration was assayed for p24Gag antigen by ELISA assay (NEN Life Science Products,Boston, MA, USA). In vitro transduction experiments weredone in 6-well plates. BMDM were transduced using 150 ng ofp24 TRIP-GFP or 300 ng of p24 TRIP-Nef vector particles,respectively/106 cells.
Intracerebral injections of viral particles were performedusing 1.5 �L of TRIP-GFP (100 ng/�L p24) and 4.3 �L ofTRIP-Nef vector particles (70 ng/�L p24).
Quantitation of gene transfer efficiency
Transduction efficiency was evaluated using flow cytometry.The mononuclear phagocyte population (BMDM) was se-lected by granularity and cell size as described (41). Aftersuitable gating, 10,000 events were collected for each exper-iment in FL1-height (488 wavelength) (CellQuest software;Becton Dickinson, Pont de Claix, France). M1 represents themean of the signal corresponding to the negative-untrans-duced macrophage population and M2 the percentage ofNef- or GFP-transduced macrophages.
Immunoprecipitation and immunoblot analysis
Primary macrophages were transduced ex vivo for 48 h. Cellextracts were precipitated in lysis buffer, loaded, denaturated,and frozen. Supernatants were collected, concentrated (Cen-triplus, Amicon, Millipore, Yvelines, France), and treated asdescribed above. Protein concentration was determined usingBCA reagent kit (Pierce, Rockford, IL, USA). For eachsample, proteins (50 �g) were fractionated on 16% SDS-PAGE gradient gels under nonreducing conditions and trans-ferred to a cellulose membrane. Nef was labeled using Nefantibody (Hybridolab, Pasteur), followed by a secondaryanti-mouse horseradish peroxidase. Proteins were detectedwith the chemiluminescence detection Kit (Amersham, Bio-sciences UK Limited, Little Chalfont, England, UK).
Intracerebral macrophage transplantation
Autologous engraftments were performed using BMDMtransplanted into the brain of Long Evans rats (n�24). Maleadult rats (250–300 g) were anesthetized with sodium pento-barbital (50 mg/kg; Sanofi, France) and positioned onto astereotaxic frame as described (41). Holes were drilled intothe appropriate locations and a 10 �L Hamilton syringe wasused to inject the cellular suspension or the vector particlesinto the hippocampus (AP–2.6 and –4.2, L1.4 and 3,DV–3.3 and –3.5) at a slow time course (0.2 �L/min). Atcessation of the injection, the needle was left in place for 15min before withdrawal. Four different groups of animals weregrafted bilaterally: 1) six rats with Nef-transduced macro-phages and, as a control, six rats with GFP-transduced BMDM,2) six rats injected with TRIP-Nef vector particles; as a control,six rats injected with TRIP-GFP vector particles. One animalinjected with TRIP-Nef vector particles died during surgery.Animals were killed 2 months after cell transplantation orvector particle injection. Rats were perfused intracardiallywith 4% paraformaldehyde (PFA). Brains were removed,
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dipped in 20% sucrose solution, frozen in Tissue-tek OCT(Miles, Elkhart, IN, USA), and stored at –80°C.
Immunocytochemical procedures
In vitro, BMDM cells were washed, fixed with 4% PFA solutionfor 20 min at room temperature and rinsed with PBS. Cellswere then treated with a blocking solution (10% goat serumand 0.2% Triton X-100 in PBS) and incubated with Nefantibody (Hybridolab, Pasteur). Next, the secondary antibodyFITC-conjugated goat anti-mouse IgG (Beckman Coulter,Roissy, France) diluted 1:500 in PBS was added to the cells.The glass coverslips were mounted in fluosaved reagent(Calbiochem, Meudon, France) and stored at –20°C. TheGFP-autofluorescent and FITC labelings were analyzed byoptical microscope (Zeiss, le Pecq, France).
For in situ immunohistochemistry, coronal OCT-coatedslices (cryostat, 25 �m) were rehydrated in PBS and incu-bated in 0.3% hydrogen peroxide in methanol for 20 min atroom temperature in a moist chamber to inhibit endogenousperoxidase. Slices were then treated with a blocking solutioncontaining 10% goat serum and 0.2% Triton X-100 in PBS.To characterize the macrophages, cells were incubated withmouse IgG anti-rat Mac-1 (Serotec, Oxford, UK, diluted 1:100in blocking solution) (47, 48). Antibodies specific for theastrocytic marker GFAP (Chemicon, Paris, France; diluted1:400 in blocking solution) and TNF-� (Chemicon; diluted1:40) were used to detect inflammatory process (49, 50).Next, a secondary biotinylated antibody (Amersham, France)was amplified by a streptavidin complex labeled with alkalinephosphatase (Dako, Cambridgeshire, UK). Slices were coun-terstained in a Mayer’s hematoxylin solution (Sigma, St.Louis, MO, USA) and analyzed by optical microscopy.
TUNEL assay
DNA strand breaks of apoptotic cells were identified in situ bythe ApopTag kit (Intergen Compagny, Oxford, UK). Braincryosections were treated with hydrogen peroxide (0.3%) inPBS for 5 min at room temperature to quench endogenousperoxidase. Slices were rehydrated with the equilibrationbuffer and incubated with the working strength TdT-basedenzymatic solution laced with nucleotides for 1 h in ahumidified chamber. The reaction was stopped using theworking strength stop/wash buffer. Slices were counter-stained with propidium iodide (1 mg/mL) and mountedunder a glass coverslip. Pictures were obtained on opticalmicroscope.
Behavioral methods
Apparatus
Open field A white circular open field (110 cm in diameterand 35 cm height) was illuminated by two indirect 60W bulbssuspended on both side walls. Four different objects wereplaced into the open field and salient visual and auditory cueswere placed in the environment to allow spatial orientation. Acamera fixed to the ceiling above the apparatus was con-nected to a videotrack system (View-point, Lyon, France),allowing the experimenter to record and observe behaviorout of the sight of the animals.
Cross maze As described in ref 51, the apparatus was madeof four arms designing a cross (total size 1 m�78 cm)connecting a central platform (Fig. 1A). cup containingsucrose pellets was presented at the end of the north arm ofthe maze. The experimental room contained a few extra-maze cues and was illuminated by two indirect 60W bulbs.
Experimental design and testing procedure
Open-field The rats were daily manipulated for 10 min for2 wk before surgery and 1 more wk during postoperativerecovery. They were subjected to the open field test 28 daysafter grafting. The general procedure has been described indetail elsewhere (52). Briefly, rats were subjected to fivesuccessive 6 min sessions separated by a 3 min delay duringwhich they were returned to their home cage. During session1, rats explored an empty environment; for sessions 2–4, theirenvironment contained four objects (A–D). During session 5,the spatial arrangement of the objects was altered to test thereaction-to-spatial change. The location of objects B and Dwas modified so that D replaced B and B was displaced at theperiphery of the arena. To neutralize the possible effects ofolfactory cues, the experimenter used plastic gloves whenmoving and manipulating the objects. The floor was cleanedwith water between each session.
Cross maze This experimental procedure (51) started 38days after surgery. Animals were food deprived until theirweight was adjusted at 85% of normal body weight. Allanimals had access to water ad libitum. Place learning startedonce the rats had become acclimated to the maze (i.e., ratsconsumed food pellets in the maze for a maximum of 2 min).Two rats from macrophage group (one from GFP and onefrom Nef condition) were discarded because they lacked foodmotivation.
Each rat received three trials a day. Between each trial ratswere returned to a waiting cage by the time another rat raninto the maze. The starting arm was either at the west or eastarm and randomly chosen, so that on 2 consecutive days everyrat started half of the time by the east arm and half of the timeby the west arm. The south arm was neither baited nor astarting point but contained a food cup with unreachablefood in order to avoid odor guidance. The time to reach thefood goal was recorded. Once the rat began to eat, the timerwas stopped and the animal was allowed to consume the foodfor 10 to 15 s. If a rat failed to reach the goal in 2 min, it wasreturned to a waiting cage.
After 7 days of learning, all rats reached the criterion (i.e.,making no visit to an unbaited arm for 2 consecutive days; 6trials). After a rat reached the criterion, it was submitted to anorientation test consisting of reaching the goal from modifiedstarting points. In the modified configuration, the position ofthe goal arm and distal environmental cues remained un-changed (Fig. 1B).
Figure 1. Cross maze apparatus. A) Learning phase. The foodgoal is presented at the end of the north arm (i.e., goal arm)of the maze. In the south arm, sucrose pellets were presentbut not available. Animals learn to locate the food goal frompseudo-random departures distributed between east and westarms. B) Orientation test. East, south, and west arms weremoved so that the angular relationships they had with thegoal arm were modified. The goal arm remained in the sameplace. To locate the food goal in this modified configuration,animals have to orient themselves using external cues.
1853Nef-INDUCED NEUROPATHOLOGY
Data collection and analysis
Open-field The video track software collected automati-cally locomotor activity in terms of distance and exploratoryactivity in terms of time of entry in areas containing objects.
Locomotor activity was compared between groups using a ttest during the first session before introduction of objects.
Times spent in areas containing each object were averagedacross individuals per group. A repeated measure ANOVA forpaired comparison (Statview program) was carried out. Themain factors were “G” as the between-subject measure and“session” as the within-subject measure. To ascertain whetherrats showed habituation, we compared sessions 2, 3, and 4.
Reaction-to-spatial-change was compared between groupsusing a t test performed on the difference between the timespent investigating the exchanged objects and the nonex-changed ones: session 5 minus session 4.
Cross maze In this task, the variable was the time to reachthe goal. We carried out repeated measures of variance toascertain the effects of the main factors and t tests for post hocanalysis.
During learning, session was the within-subject factor andgroup the between-subject factor.
In the orientation test, group was the between-subjectfactor and “condition” was the within-subject factor with twolevels, standard configuration vs. test.
RESULTS
Highly efficient gene transfer into primary cultures ofbone marrow-derived macrophages
Nondividing terminally differentiated BMDM weretransduced with TRIP vector deleted in the U3 regionof the LTR and expressing the HIV-1 nef gene underthe control of the CMV internal promoter. Fifteen daysafter transduction, FACS analysis was performed toassess gene transfer in the BMDM (Fig. 2A). We ob-served a signal showing that 70% of macrophagesexpressed Nef (M2) compared with untransducedBMDM (M1) (Fig. 2B). This highly efficient transduc-tion was obtained using a concentration of TRIP-Nefvector particles corresponding to 300 ng of p24 (seeMaterials and Methods). A control of transduction wasassessed using GFP lentiviral particles at a concentra-tion of 150 ng of p24. As shown in Fig. 2C, 75% ofBMDM was efficiently transduced with TRIP-GFP vector(M2) compared with untransduced cells (M1). Thesustained Nef expression remains stable for at least 1month in vitro without any cytotoxic effect noted in theBMDM culture (data not shown).
Characterization of transduced cells
Primary culture of BMDM (Fig. 3A) was characterizedby immunohistochemical procedure using Nef anti-body or autofluorescence detection for GFP. As shownin Fig. 3B, after 15 days of transduction the greatest partof BMDM expressed Nef. The same result was obtainedusing TRIP-GFP vector as a control of transduction(Fig. 3C). All BMDM came from the mononuclear
phagocyte lineage as shown using ED1 and Mac-1antibodies (41).
Western blot analysis was performed under nonde-naturating conditions on cellular extracts and superna-tants of BMDM transduced with the lentiviral TRIPvector expressing Nef or GFP. In the cellular extracts ofNef-transduced BMDM (Fig. 3D, line b), Nef can bedetected using a monoclonal antibody as a 27 kDaprotein. The same antibody was unable to reveal Nefexpression in the concentrated supernatant of Nef-transduced BMDM (Fig. 3D, line c). No signal wasobserved in the cellular extract of GFP-transducedBMDM (Fig. 3D, line a).
Viability of transduced macrophages transplanted intothe rat hippocampus
The high efficiency of Nef primary macrophages trans-duction allowed us to investigate the effect of Nef in therat brain, so we performed engraftments of Nef-trans-duced BMDM into the hippocampus area (Fig. 4A).
We observed that Nef was detected around theinjection site of transplanted Nef-BMDM 2 monthsafter the graft (Fig. 4B). Similar results were obtainedwith GFP-transduced BMDM (Fig. 4C). Analysis ofdirect vector particles injection showed that Nef or GFPparticles surrounded the needle track in the hippocam-pus (Fig. 4D, E, respectively).
Figure 2. TRIP-Nef efficient transduction of BMDM. A) Stablecell size and granularity dot plot showing the selected regioncorresponding to the BMDM cell population analyzed byFACS. These parameters were identical all through the ex-periment for every conditions (untransduced cells, TRIP-NefBMDM, TRIP-GFP BMDM). B) The pick signal (M2) showsthat 70% of the macrophages expressed Nef. C) Controlmeasures were assessed by TRIP-GFP transduction (M2). Bothtransductions were compared with untransduced cells (M1).
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Chemotactic and neurotoxic effects of transplantedNef-BMDM in the rat hippocampus
Two months after the graft, transduced Nef and GFPBMDM were still alive at the injection site and could bedetected using Mac-1 antibody (Fig. 5A). The presenceof Mac-1-positive cells around the injection site ofNef-transduced-BMDM (Fig. 5A, bottom) suggests therecruitment of peripheral or resident monocytes/ mac-rophages as a consequence of Nef expression. Similarresults were observed around the injection sites of Nefviral particles (data not shown). By contrast, in thebrain of rats grafted with GFP-transduced BMDM, onlygrafted macrophages were stained with Mac-1 antibody(Fig. 5A, top).
We examined each transplanted and injected brainslice for astrocytes reactivity to assess local astrogliosis
using GFAP astrocytic marker. Two months after thegraft, enhanced GFAP staining was detected around theNef-transduced BMDM (Fig. 5B, bottom) whereas nosignificant sign of astrogliosis was seen around the graftof GFP positive BMDM (Fig. 5B, top).
As brain damage can be induced by direct or indirectmechanisms involving proinflammatory cytokines, weexamined the presence of TNF-� expression in the ratbrain. We observed about the Nef-transduced BMDM asignificant signal of TNF-� 2 months after the engraft-ment (Fig. 5C, bottom). Moreover, TNF-� expressionwas detected around the astrogliosis only in the case ofNef-BMDM graft. No significant astrogliosis or TNF-�expression was detected around the GFP-BMDM graft(Fig. 5C, top).
The next step was to analyze whether Nef couldinduce an apoptotic effect in the rat brain as a conse-quence of the inflammatory process. Figure 5D illus-trates that no apoptosis corpus was observed 2 monthsafter Nef-BMDM or GFP-BMDM transplantation intothe rat hippocampus.
Behavioral results
Exploratory activity
Statistical ANOVA for exploratory activity is summa-rized in Table 1. It shows no statistical differencebetween groups (macrophages GFP vs. macrophagesNef and particles GFP vs. particles Nef) for locomotoractivity and habituation, although there is a tendencyfor animals receiving macrophages expressing Nef toexhibit a higher level of exploratory activity toward theobjects (illustrated in Fig. 6). We cannot exclude thislack of statistical difference between groups as anexpression of a high variability between Nef individuals.There is no significant interaction group X session,indicating a similar habituation in both groups.
However, the reaction-to-spatial-change is signifi-cantly different for the GFP and Nef animals of theparticle group (time of object exploration for S5-S4,t�0.04), and nearly reach significance for the GFP and
Figure 3. Characterization of transduced BMDM. A) Typicalshape of primary macrophages in culture. B) BMDM trans-duced with TRIP-Nef vector and labeled with a specificantibody. C) GFP BMDM observed by autofluorescence.D) Detection of Nef by Western blot; GFP BMDM extracts(line a), Nef BMDM extracts (line b), Nef BMDM cell culturesupernatant (line c).
Figure 4. Intracerebral Injections. A) Sche-matic illustration of anatomical localizationof the injections performed in the hip-pocampus. Histological results of Nef (B, D)and GFP (C, E) 2 months after cell orparticle grafts.
1855Nef-INDUCED NEUROPATHOLOGY
Nef animals of the macrophage group (time of objectexploration for S5-S4, t�0.054).
Place learning
Statistical analyses of variance for place learning andorientation test are summarized in Table 2.
Statistical analyses conducted on “time to reach thegoal” showed a main effect of session, no group effectand no significant group X session interaction, indicat-ing that all animals learned the task in 7 days or less anddid not differ in the time needed to reach the goal asillustrated in Fig. 7A, B.
Orientation challenge
We compared the time to reach the goal for GFP andNef animals (group effect) before modification of thespatial arrangement of the maze arms (performance atthe 7th day of training) and during the orientation testfor which configuration of the apparatus is modified(condition effect). Statistical results of the repeatedmeasures of variance for the main effects are given inTable 2. As illustrated in Fig. 7C, D, these results suggestthat the orientation test did not affect the performanceof both groups in the same way.
In the macrophage group, post hoc analysis revealed
a significant condition effect for group 2 (Nef) (t�2.8,ddl�4, P�0.048) but not for group 1 (GFP) (t�1 , NS).The time to find the goal for the Nef group was longerduring the orientation test than during the learningcondition, but remained stable for the GFP animalswhatever the configuration of the maze.
In the particle groups, post hoc analysis showed acondition effect for group 2 (Nef) (t�3.24, ddl�4,P�0.03) but not for group 1 (GFP) (t�1 , NS).
Overall, these results show that only Nef animals(macrophage and particle) spent significantly moretime reaching the goal in the modified configurationthan in the standard configuration, suggesting that theability to adapt their behavior to sudden configuralchanges is altered in Nef animals compared with GFPanimals.
DISCUSSION
In this report we provide the first evidence that thegraft of Nef-transduced BMDM into the rat brain caninduce an early observable neuropathology (i.e., in-flammatory effects associated with cognitive defects.
Our in vitro results demonstrate that HIV-1 lentiviralvector allowed high and stable expression of Nef intransduced macrophages and correct maturation and
Figure 5. Histological analyses of Nef expres-sion in the rat hippocampus. Comparativeanalysis of Nef (bottom) and GFP (top)BMDM graft. A) Detection of monocytes/macrophages by Mac-1 in and around thetransplant. B) Detection of local astrogliosisaround the Nef BMDM graft using GFAPmarker compared with GFP BMDM. C) Ex-pression of TNF-� around the astrogliosis inNef graft. D) Absence of apoptosis events inboth graft conditions. Black bars indicatethe few apoptotic cells found in the slice.
TABLE 1. Statistical analyses for exploratory activitya
Behavior Macrophages Particles
Locomotor activity For session 1: Group effect, F(1–10) � 1, NS For session 1: Group effect, F(1–9) � 1, NSHabituation Group effect, F(1–10) � 4.2, NS Group effect, F(1–9) � 1, NS
Session effect, F(2–20) � 14.34, P � 0.0001* Session effect, F(2–18) � 3.16, P � 0.067Group X session, F(2–20) � 1, NS Group X session, F(2–18) � 1, 2, NS
Reaction to spatial change(S5–S4) Group effect, t � 0.054 Group effect, t � 0.04
a Nef and GFP animals displayed similar locomotor activity and a normal habituation of exploratory activity toward objects. For habituation,a slight tendency of higher level of exploratory activity can be observed in Nef groups. Only GFP animals reacted to the spatial change byrenewing exploration toward objects during session 5 vs. session 4.
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expression of Nef in the BMDM. Moreover, neithercytotoxic effects nor secretion of Nef protein from thecell was observed in the culture over 1 month aftertransduction. This suggests that our in vivo observationsare unlikely to result from the effect of the secretedprotein but rather from the expression of Nef by themacrophages.
In vivo, we grafted BMDM expressing Nef into thehippocampus area to 1) detect HIV-1-gene sequences(in particular, gag and nef ) into the hippocampus (53),and behavioral effects of hippocampal damage in therat are described in the literature (see ref 54 for review)and because 2) earlier studies have described thecentral role of Nef in toxic mechanisms, notably by
modulating the intracellular machinery of the host cell(55–57). It has been shown in vivo that HIV neuropa-thology may be correlated with the presence of Nefexpressed in astrocytes (26, 27, 53). 3) We developed anefficient transduction method for targeting originalHIV cells (macrophages) using a TRIP vector (41).
To mimic the human HIV neuropathology in ananimal model, we grafted onto the rat hippocampusmacrophages ex vivo transduced with TRIP-Nef vector.
One month after grafting, we began the investigationof behavioral functions in transplanted animals by firstconducting open field exploratory behavior. Our re-sults showed that reaction to spatial change is affectedin animals receiving Nef. Indeed, we did not observe
Figure 6. Exploratory behavior to-ward objects. A) Locomotor activityand habituation. The top graphshows the locomotor activity exhib-ited in the empty open field (ses-sion 1) by macrophage and particlerats. No difference was observedbetween GFP (white) and Nef(black) groups. The bottom graphsshow habituation of exploratory ac-tivity toward the objects during ses-sions 2–4. For particle (left) andmacrophage (right) groups, GFP(open circle) and Nef (blacksquare) rats exhibited similar timeof contact with objects over ses-sions. B) Reaction to spatial changefor particle (left) and macrophage(right) groups, GFP animals dis-played a higher level of explorationfor session 5 than for session 4,exhibiting a normal reaction tospatial change. The reaction to spa-tial change is not observed for Nefanimals.
TABLE 2. Main statistical effects for place learning and orientation testa
Behavior Macrophages Particles
Learning Group effect, F(1–8) � 1, NS Group effect, F(1–9) � 1, NSSession effect, F(6–48) � 9.24, P � 0.00* Session effect, F(6–54) � 8.0, P � 0.00*Group X session, F � 1, NS Group X session, F � 1, NS
Orientation test Group effect, F(1–8) � 3.961, P � 0.083 Group effect, F(1–9) � 12.03, P � 0.007*Condition effect, F(1–8) � 5.71, P � 0.044* Condition effect, F(1–9) � 10.49, P � 0.01*Group X condition, F(1–8) � 4.05, P � 0.079 Group X condition, F(1–9) � 8.62, P � 0.016*
a For place learning, we observed a main effect of “session” for all groups, no “group” effect, and no significant interaction group X Session,suggesting that the learning time course is identical for all animals. For orientation test, we observed a main “condition” effect, indicating thatmodification of the shape of the maze induced an increase in the time to reach the goal. Post hoc tests are given in the text.
1857Nef-INDUCED NEUROPATHOLOGY
increased locomotor activity or impaired habituation ofexploratory activity as would be expected if the hip-pocampus was lesioned (58, 59). However, the reactionto spatial change is impaired in Nef animals, suggestingthat spatial memory is affected in these rats. The abilityto memorize a spatial configuration is known to rely onhippocampal function in animals (60–62) and humans(63, 64). Although supporting spatial representation isa main characteristic of the hippocampus (65), twoprimary reasons, not mutually exclusive, can explainthis incomplete effect.
First, the construction of spatial representation is oneof the major functions in rodents. Thus, it is supportedby the functioning of several interconnected brainareas including the prefrontal and parietal cortices, theseptum, CA1 and CA3 hippocampus, and the amygdala(54, 61). Such a redundant function is unlikely to beaffected by a minor lesion of one of the targeted area,as may have been the case only 1 month after grafting.Our histological results indicated only focal inflamma-tion of the hippocampus around the graft (Fig. 5A)whereas earlier work showed that complete lesion orinactivation of the hippocampus is necessary to inducerobust impairment in place representation (58). More-over, the Nef transgenic mouse model described byHanna and colleagues (30) supports the view that acertain level of expression of the transgene is necessaryto achieve sufficient protein synthesis to induce anAIDS-like pathogenicity (30).
Second, the open field test might have been carriedout too early for the hippocampus to be fully affectedby Nef expression. This explanation is supported by the
observation that the performance is quite variableamong rats receiving Nef. This variability may be due toindividual factors in Nef action within the hippocam-pus over time. This hypothesis is supported by thedrastic behavioral impairments observed in the spatiallearning task.
In the spatial learning experiment, animals receivingparticles or macrophages expressing Nef showed nor-mal spatial learning of the food goal location. Animalsreceiving the control protein GFP and those receivingNef demonstrated significant decreases in the timeneeded to reach the food, suggesting that all animalslearn to locate the goal progressively. However, severalstudies have shown that rats can locate spatial goals bycomputing intra- and extra-maze cues and that thehippocampus participates crucially in the integration ofdistal configural cues (51, 62, 66).
It is known that spatial tasks favor in rodents thedevelopment of strategies that affect learning processes(67–70). Animals spontaneously display place strategiesthat were reinforced in our protocol (51). During theorientation test, the angular relationships between thearms of the maze were altered such that the armcontaining the food goal was the only place havingstable spatial relationships with the external environ-ment. Our results for this test showed that GFP rats stillperformed as efficiently as in the standard configura-tion, suggesting they were able to use a memorizedconfiguration of the goal based on distal landmarks.On the contrary, Nef animals (for either the macro-phage or particle groups) showed a marked impair-ment, as for these groups the time needed to reach the
Figure 7. Spatial learning of food location and orientation test. Top graphs show learning curves for particle (A) andmacrophage (B) groups over daily sessions. GFP (open circle) and Nef (black square) rats learn the task to a similar extent.Bottom graphs show the time to reach the goal during the orientation test. Nef animals (particle, C; macrophage, D) increasedsignificantly the time to reach the goal in the modified configuration (test) compared with the standard configuration(learning). This change in behavior is not observed for GFP animals.
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goal significantly increased after alteration of angularrelationships between the arms of the maze. We previ-ously showed that in this configuration, normal animalssolve the task using primarily extra-maze cues, leavingintra-maze cues irrelevant (51). The ability to adapt thebehavior to sudden configural changes based on spatialstrategy implies using and updating the spatial relation-ships between the components of the environment.These cognitive abilities, known to rely on the integrityof the hippocampal (58, 59, 61), are specifically alteredin Nef animals. We can therefore infer that animalsreceiving Nef exhibit cognitive defects resulting fromhippocampal dysfunction.
At the completion of behavioral experiments, ani-mals were killed and the brains were removed forhistopathological analyses. Our results showed that 2months after transplantation, the presence of Nef inthe rat hippocampus induced a significant astrogliosisand a recruitment of monocytes/macrophages aroundthe injection site of the graft. These histopathologicaleffects are unlikely to be due to a general effect of aforeign protein expressed in the brain, as GFP-BMDMdid not produce a similar astrogliosis. Our results are inaccordance with those of Koedel and colleagues (40),who demonstrated that injection of soluble HIV-1 Nefin the brain induced the recruitment of leukocytestoward the inflamed region. However, as they injecteda soluble form of the protein, they conclude that Nefrepresents the viral factor involved in HIV neuropathol-ogy. Our present data further show that not only is theprotein itself able to induce a neuropathology but that,as in infected patients, the protein expressed by theHIV target cells (i.e., macrophages) induced an earlynoticeable neurotoxicity. Indeed, it has never beenshown that Nef is secreted at a sufficient rate to be ableto induce an inflammatory process. Therefore, wehypothesize that Nef modifies the cellular machinery ofthe HIV target cells, which secondarily spreads inflam-matory processes toward the surrounding cells of thetarget area. In accordance with this hypothesis, we showa significant expression of the proinflammatory cyto-kine TNF-� specifically around the injection site of theNef grafts, an inflammatory process not observed inGFP-transduced cells. The TNF-� secretion seems to becorroborated with the localization of astrogliosis, butcould be the consequence of the expression of Nef bythe macrophages. In fact, Nef neurotoxicity has beenlinked to the production of quinolinic acid by macro-phages. The quinolinic acid, as the proinflammatorycytokine TNF-�, has been shown to be produced byactivated macrophages in several inflammatory braindiseases, including AIDS dementia complex (37, 71–73).
In the literature, HIV-1 neurotoxic proteins (i.e.,gp120 and tat) have been shown to induce apoptoticevents (74–76). It has been further suggested thatapoptosis is mediated through indirect mechanismsinvolving modifications of the signaling pathway ofinfected cells (77–81). However, the results concerningNef are controversial. Rasola and collaborators (82)concluded that Nef enhances programmed cell death,
whereas other studies have described the role of Nef inpromoting resting cell infection (83) or argued thatNef is associated with anti-apoptotic signals (84, 85). Inour study, we did not find any apoptotic events associ-ated with astrogliosis or TNF-� expression.
To conclude, our results showed that Nef, through itsexpression by HIV target cells, is one of the viral factorsthat influence the early development of a neuropathol-ogy that can produce cognitive impairments. Moreover,in accordance with the results obtained in other labo-ratories (40, 86), our data suggest that Nef may mediatechemotaxic effects and a reactive response by macro-phages and astrocytes. Our study has investigated, forthe first time, both behavioral and histopathologicaleffects of a viral HIV-1 protein expressed by the naturalHIV-1 target cells grafted onto a specific brain areaknown to be affected in HIV-demented patients. Therat model of HIV-associated neuropathology should beuseful for the study of fundamental mechanisms ofneurotoxicity exerted by a specific protein expressed bymacrophages within the brain. Furthermore, it empha-sizes the interest of correlating histopathological andbehavioral data in evolutive neuropathologies affectingcognitive functions.
E.M. was supported by a fellowship from the Pasteur-Weizmann Fundation. S.G. was supported by a postdoctoralgrant from “la Fondation pour la Recherche Medicale”(SIDAction). This work was also performed by grants fromAgence Nationale pour la Recherche contre le SIDA andl’Institut Pasteur. We would like to acknowledge Pr. Sylvie vander Werf for critical reading of the manuscript, Brian Mollesfor correcting the English and two anonymous referees forhelpful comments.
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Received for publication June 24, 2004.Accepted for publication July 26, 2004.
