brain-derived neurotrophic factor-mediated protection of striatal neurons in an excitotoxic rat...

HUMAN GENE THERAPY 10:2987– 2997 (December 10, 1999)Mary Ann Liebert, Inc.

Brain-Derived Neurotrophic Factor-Mediated Protection ofStriatal Neurons in an Excitotoxic Rat Model of Huntington’s

Disease, as Demonstrated by Adenoviral Gene Transfer

ALEXIS-PIERRE BEMELMANS,1 PHILIPPE HORELLOU,1 LAURENT PRADIER,2

ISABELLE BRUNET,1 PHILIPPE COLIN,1 and JACQUES MALLET1

ABSTRACT

Huntington’s disease (HD) is a genetic disorder leading to the degeneration of striatal GABA-ergic outputneurons. No treatment is currently available for this devastating disorder, although several neurotrophic fac-tors, including brain-derived neurotrophic factor (BDNF), have been shown to be beneficial for striatal neu-ron survival. We analyzed the effect of adenovirus-mediated transfer of the BDNF gene in a model of HD.Using a stereological procedure, three groups of rats were given an intrastriatal injection of adenovirus en-coding BDNF, b -galactosidase, or sham surgery. Two weeks after treatment, the animals were lesioned withquinolinic acid (QUIN), a toxin that induces striatal neuron death by an excitotoxic process. One month af-ter the lesion, histological study revealed that striatal neurons were protected only in rats treated with theBDNF adenovirus. Volume measurements showed that the QUIN-induced lesions were 55% smaller in theBDNF adenovirus-treated group than in the b -galactosidase adenovirus-treated group (p , 0.05), and thesham-treated group (p , 0.05). To determine the survival of striatal GABA-ergic output neurons after theQUIN-induced lesion, we immunostained brain sections with DARPP-32, an antibody specific for striatal out-put neurons. Prior treatment with the BDNF adenovirus resulted in a cell survival of 64%, whereas that af-ter b -galactosidase treatment was 46% (p , 0.05), showing that the BDNF adenovirus protected the striatalneurons. These results indicate that transfer of the BDNF gene is of therapeutic value for Huntington’s dis-ease.

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OVERVIEW SUMMARY

Huntington’s disease results from a progressive neurode-generative process affecting primarily the neostriatum. TheGABA-ergic output neurons are the most sensitive cell typein this anatomical structure. Brain-derived neurotrophicfactor (BDNF) has been shown to be a potent neurotrophicfactor for striatal GABA-ergic neurons in vitro and in vivo .We investigated whether BDNF protected striatal neuronsin a rodent model of Huntington’s disease, by testing the ef-fect of a recombinant adenovirus producing this neu-rotrophin in rats injected with quinolinic acid. BDNF wasproduced for at least 6 weeks after intrastriatal injection ofthe adenoviral vector. Most importantly, recombinantBDNF reduced the size of the quinolinic acid-induced lesion

in the striatum and protected striatal GABA-ergic outputneurons from the excitotoxic damage. Thus, in vivo BDNFgene transfer has potential for the neuroprotective treat-ment of Huntington’s disease.

INTRODUCTION

HUN TIN GTO N ’S DISEA SE (HD) is an autosomal dominant ge-netic disorder involving the neurodegeneration of primar-

ily the striatal g -aminobutyric acid (GABA)-ergic output neu-rons (i.e., medium-sized spiny neurons). This results inexcessive involuntary movements (chorea) accompanied bycognitive deficits and personality changes. In 1993, it wasshown that HD is caused by the expansion of a polym orphic

1Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, UMR C9923, Centre National de laRecherche Scientifique, Hôpital de la Pitié-Salpêtrière, Bâtiment CERVI, 83 Boulevard de l’Hôpital, 75013 Paris, France.

2GenCell Rhône-Poulenc Rorer, Centre de Recherches de Vitry-Alfortville, 94403 Vitry-sur-Seine, France.

CAG/polyglutamine repeat located in the IT15 gene (Hunting-ton’s Disease Research Collaborative Group, 1993). The pro-tein encoded by IT15, huntingtin, is present throughout the central nervous system and in peripheral tissues, but neurode-generation is restricted to striatal GABA-ergic output neurons(Sharp et al., 1995; Trottier et al., 1995). It is now establishedthat expansion of the polyglutam ine tract causes the intranu-clear aggregation of the mutant huntingtin, known as neuronalintranuclear inclusion (Davies et al., 1997; Ordway et al., 1997).Inclusions are found in the brains of both patients (DiFiglia etal., 1997) and transgenic mice bearing the mutated HD gene(Davies et al., 1997), but their contribution to the pathogenic-ity of the mutant huntingtin is unclear (Saudou et al., 1998;Sisodia, 1998). Nevertheless, in two studies, it has been sug-gested that the expanded polyglutamine repeat-induced celldeath might be due to activation of caspases, a family ofproapoptotic proteins (Ona et al., 1999; Sanchez et al., 1999).Although these findings constitute progress toward the eluci-dation of the pathological mechanism, they have not yet led toany obvious treatment, and HD remains an incurable disease.

Several strategies are currently being explored to developtreatments for HD, including both restorative and preventiveapproaches. The restorative strategy involves the intracerebralgrafting of fetal striatal cells and is undergoing clinical trials inFrance and the United States (Horellou and Mallet, 1998;Kopyov et al., 1998). The developm ent of a preventive treat-ment to protect neuronal cells from the neurodegenerativeprocess would be of great value for a clinical application bypreventing the onset of symptoms. Animal models that allowthe testing of possible treatments have been developed. Theseare based on excitotoxic cell death, which is thought to be in-volved in the pathological developm ent of HD. Thus, severalmodels involve the use of excitotoxins to induce neuronal celldeath mimicking the pattern of neurodegeneration observed inHD (DiFiglia, 1990). Quinolinic acid (QUIN) has been widelyused in models of HD. QUIN acts on the N-methyl-D-aspartate(NMDA) receptor and if injected intrastriatally, it reproducesthe cell loss and neurochemical deficits of HD (Beal et al.,1986). In this model of HD, various neurotrophic factors havebeen shown to be effective for the protection of striatal GABA-ergic output neurons. Nerve growth factor (NGF) (Martinez-Serrano and Björklund, 1996), ciliary neurotrophic factor(CNTF) (Anderson et al., 1996; Emerich et al., 1997), and glialcell line-derived neurotrophic factor (GDNF) (Araujo and Hilt,1997) protect striatal GABA-ergic neurons from excitotoxic celldeath in vivo . Several observations have suggested that brain-derived neurotrophic factor (BDNF), a member of the neu-rotrophin family, protects striatal GABA-ergic output neurons.In striatal primary culture, BDNF increases the survival, somasize, and branching point number of GABA-expressing neurons(Mizuno et al., 1994; Widmer and Hefti, 1994; Ventimiglia etal., 1995). The neuroprotective effect of BDNF has also beendemonstrated in two in vitro models of HD: first, BDNF pro-tects striatal cells exposed to an excitotoxic stress in culture(Nakao et al., 1995); second, BDNF abolishes the apoptosis ob-served in striatal cells transfected with the coding sequence ofthe mutant huntingtin (Saudou et al., 1998). Thus, determiningthe protective potential of BDNF in in vivo models of HD is ofmajor importance.

In this study, we tested the effect of BDNF, using a recom -binant adenovirus. This vector was injected into the striatum ofrats. Lesions were then produced 2 weeks later in the same areawith QUIN. The adenovirus-medi ated transfer of the BDNFgene led to the strong expression of this neurotrophin gene inthe rodent striatum, and the recombinant BDNF protected thestriatal GABA-ergic output neurons from excitotoxic cell deathinduced by QUIN.

MATERIALS AND METHODS

In vitro adenoviral gene transfer

Ad-BDNF and Ad- b Gal stocks were obtained as previouslydescribed (Stratford-Perr icaudet et al., 1992; Gimenez y Ribottaet al., 1997). For in vitro infection, HeLa cells were plated ata density of 5 3 105 cells per well in six-well culture plates andinfected as described below. The culture medium (Dulbecco’smodified Eagle’s medium containing 10% fetal calf serum) wasremoved and cells were infected with either Ad-BDNF or Ad-b Gal at a multiplicity of infection (MOI) ranging from 20 to80 plaque-forming units (PFU) per cell in 0.5 ml of serum-freemedium for 1 hr. Afterward, the culture medium was addedback to the cells. Three days later, cells were extensively rinsedwith phosphate-buffer ed saline (PBS) and were then incubatedfor 24 hr in fresh medium. The amount of BDNF in the culturemedium was determ ined with the BDNF Emax ImmunoAssaysystem (Promega, Madison, WI) according to the manufacturerinstructions. Primary striatal cultures were obtained by dissec-tion of embryonic day 14 (E14) rat fetus lateral ganglionic em-inences and subsequent culture under serum-free conditions, aspreviously described (Barkats et al., 1996). This method of cul-ture allows purified neurons ( . 95%) to be obtained, as demon-strated by immunocytochem istry against glial fibrillary acidicprotein and neurofilament (Weiss et al., 1986). The primarycells were plated in 12-well culture plates at a density of 2 3105 cells per well and incubated for 3 days. They were then in-fected at an MOI of 100 PFU per cell as described for HeLacells. Four days after infection, cells were fixed for 20 min with4% paraformaldehy de (PFA) in PBS. Immunologica l detectionof calbindin was performed as described below (see Histolog-ical Procedures). The anti-calbindin monoclonal antibody(Sigma, Saint Quentin Fallavier, France) was used at a 1:5000dilution. For each well, calbindin-positive cells were countedin 12 random ly selected microscope fields.

In vivo adenoviral gene transfer in a rat model of HD

Recombinant adenoviruses were diluted in PBS. FemaleSprague-Dawley rats (Charles River, Saint Aubin les Elbeuf,France), under ketamine–xylazine anesthesia, were injected in theleft striatum with a total amount of 5 3 107 PFU (0.25 m l/min)in three equal aliquots of 1 m l each in the head of the caudateputamen, at the following coordinates: 1.2 mm anterior to bregma;2.5 mm lateral to bregma; 5, 4.6, and 4.2 mm ventral to the duralsurface. Sham-treated rats were injected with PBS only. Twoweeks later, QUIN (120 nmol in 2 m l of PBS) was injected at arate of 0.4 m l/min at the same site (1.2 mm anterior to bregma,2.5 mm lateral to bregma; 4.6 mm ventral to the dural surface).

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Quantification of BDNF in vivo

BDNF levels were determined in striatal lysates from rats in-jected as described above. Rats were sacrificed by chloral hy-drate injection, and striata were dissected and homogenized at4°C in 500 m l of a lysis buffer containing 137 mM NaCl, 20mM Tris (pH 7.5), 1% Nonidet P-40 (NP-40), 10% glycerol, 1mM phenylmethylsul fonyl fluoride (PMSF), aprotinin (10m g/ml leupeptin (1 m g/ml), and 0.5 mM vanadate. After 15 mincentrifugation at 13,000 3 g, BDNF was measured in the su-pernatant using the BDNF Emax ImmunoAssay system(Promega, Charbonniè res, France).

Histological procedures

Four weeks after the QUIN lesion, the rats were killed bydeep anesthesia with chloral hydrate and transcardial perfusionwith 100 ml of 0.9% NaCl, followed by 250 ml of ice-cold 4%PFA in PBS. Brains were postfixed in the same fixative for 1hr and were cryoprotected by immersion in 15% saccharose dis-solved in PBS. Brains were then cut into 16- m m-thick coronalsections with a cryostat and were stored at 2 80°C.

For in situ hybridization, an oligonucleotide probe comple-mentary to the coding sequence of the FLAG peptide (Brizzardet al., 1994) was synthesized (5 9 -CTT CTA GTC ATC GTCGTC CTT GTA GTC GCC-3 9 ) and radioactively labeled using [35S]dATP and terminal deoxynucleotidyl transferase(Promega) according to the manufacturer instructions. For hy-bridization, sections were dehydrated and incubated overnightat 42°C in 50% formamide, 1 3 Denhardt’ s solution, 4 3 SSC(1 3 SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 100mM dithiothreitol, 10% dextran sulfate, Escherichia coli tRNA(250 m g/ml), poly(A) 1 RNA (250 m g/ml), salmon sperm DNA(250 m g/ml), and 0.2 pmol of labeled oligonucleotide per sec-tion. Sections were then rinsed twice in 1 3 SSC for 15 min at53°C, twice in 0.5 3 SSC for 15 min at 53°C, and once in 0.5 3

SSC for 15 min at room temperature. They were then dehy-drated and dipped in Ilford K5 emulsion. Sections were exposedfor 2 months, then developed in Kodak (Rochester, NY) D19developer, lightly counterstained with cresyl violet, andmounted.

b -Galactosidase activity was detected enzymatically by in-cubating the sections with 5-bromo-4-chlor o-3-indolyl- b -D -galactosidase (X-Gal) as previously described (Barkats et al.,1996). After 2 hr of incubation, the sections were counterstainedwith neutral red and mounted.

For immunohistoc hemical detection of DARPP-32, endoge-nous peroxidase activity in the sections was quenched with0.3% H2O2 in methanol. Subsequently, sections were preincu-bated in PBS containing 10% normal horse serum. Mouse mon-oclonal anti-DARPP-32 antibody (generously donated by Drs.Greengard and Hemmings) was diluted 1:20,000 in the prein-cubation solution. The sections were incubated overnight atroom temperature, and the primary antibody was detected us-ing the Vectastain Elite ABC kit (Vector Laboratories,Burlingame, CA) according to the manufacturer instructions.Peroxidase activity was detected with Vector SG substrate kit(Vector Laboratories).

Lesion volume measurement and cell counting

Lesion volume and striatal volume were determined by cam-era lucida drawings of DARPP-32-immunostain ed sectionstaken every 400 m m from 2.2 mm anterior to bregma to 1.0 mmposterior to bregm a.

The lesion and striatal areas of each section were integratedto obtain lesion and striatal volum es. Cells were counted onthree sections located at the anteroposterior level, where the le-sion area was maximal. Using the PC-based Biocom (Les Ulis,France) software coupled to a Leitz (Wetzlar, Germany) mi-croscope (Hirsch et al., 1992), ipsi- and contralateral striata

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FIG. 1. Map of the adenoviral constructs. Escherichia coli lacZ and rat BDNF genes are under the transcriptional control ofthe Rous sarcoma virus long terminal repeat promoter. The rat BDNF gene is fused with the coding sequence (30 base pairs) ofthe FLAG peptide. BDNF and FLAG are separated by a glycine codon.

were encircled through a 3 4 objective, and, in this defined area,DARPP-32-positive cells were counted in 15 randomly selectedsquare fields of 4 3 104 m m2 through a 3 10 objective. Thisenabled the total number of cells to be deduced from the mea-surem ents of area and cell density.

Statistical analysis

All values are expressed as means 6 SEM. Groups werecompared with StatView software (BrainPower, Calabasas,CA) by one-way analysis of variance (ANOVA) follow ed bythe Scheffé post hoc test, except for the data in Fig. 6A and B,for which an unpaired Student t test was used. In all analyses,the null hypothesis was rejected at the 0.05 level.

RESULTS

Recombinant adenovirus harboring BDNF produces afunctional protein

The recombinant vectors used in this study, Ad-BDNF andAd- b Gal, were derived from a human type 5 adenovirus withthe E1 and E3 regions deleted. They encode rat BDNF and E.coli b -galactosidase, respectively. The BDNF cDNA was in-serted upstream from the coding sequence of FLAG (Brizzardet al., 1994). This construct yields a BDNF–FLAG fusion pro-tein, which made it possible to distinguish the recombinantBDNF from the endogenous BDNF (Fig. 1).

We investigated whether Ad-BDNF directed the synthesis ofthe BDNF protein by infecting HeLa cells with various dosesof Ad-BDNF. Four days after infection, the amount of BDNFin culture medium was determined by enzyme-linked im-munosorbent assay (ELISA). The smallest MOI used, 20plaque-forming units (PFU) per cell, was sufficient for BDNFto be detected. Moreover, the amount of BDNF increased lin-early with the MOI (r2 5 0.975; p 5 0.0001), up to 18.5 ng of

BDNF/5 3 105 cells/day for 80 PFU per cell, the highest MOItested (Fig. 2A).

We next assayed the biological activity of the recombinantBDNF. Primary striatal cultures obtained by dissection of thelateral ganglionic eminence of E14 rat fetuses were infectedwith either Ad-BDNF or Ad- b Gal on day 3 (after plating). Theywere fixed on day 7 and processed for calbindin immunohisto-chemistry. Calbindin-positiv e cells were counted and twice asmany such cells were detected in the Ad-BDNF-infected cul-tures as in the Ad- b Gal-infected or noninfected cultures (Fig.2B).

Experimental paradigm of the in vivo study

We tested the neuroprotective effect of BDNF in an excito-toxic rodent model of HD. Female Sprague-Daw ley rats wereassigned to three groups: Ad-BDNF treatment (n 5 10), Ad-b Gal treatment (n 5 8), and sham treatm ent (n 5 8). As de-scribed in Materials and Methods, each rat received a stereo-taxic injection in the neostriatum of Ad-BDNF, Ad- b Gal, orvehicle (PBS). Two weeks after treatment, two rats from theAd-BDNF group were killed for evaluation of recombinantBDNF expression. At the same time, the remaining animals re-ceived a QUIN injection (120 nmol) in the neostriatum. Fourweeks later, the animals were killed and the lesion was studiedhistologically.

In vivo expression of recombinant BDNF

In vivo expression of the BDNF transgene was analyzed byin situ hybridization 6 weeks after Ad-BDNF injection. An an-tisense oligonucleotide probe directed against the FLAG pep-tide-coding sequence detected the BDNF–FLAG transcript in alarge region of the neostriatum , surrounding the injection site(Fig. 3B and D). The BDNF–FLAG transcript was also detectedin the corpus callosum, mimicking the pattern of b -galactosi-dase staining in Ad- b Gal-treated rats (Fig. 3E and F). Expres-


FIG. 2. In vitro expression of recombinant BDNF. (A) BDNF amount in the culture medium 3 days after infection of HeLacells with various doses of Ad-BDNF. The quantity of BDNF detected increased linearly with MOI (r2 5 0.975, p 5 0.0001).(B) Effect of recom binant BDNF release on calbindin-positive cells after Ad-BDNF infection of primary striatal cultures. Fourdays after the infection with Ad- b Gal or Ad-BDNF of primary striatal cells from sister cultures, calbindin-positive cells were de-tected. Values are means for total calbindin-positive cell counts determined for each well on 12 randomly selected microscopefields through a 3 10 objective. Similar results were obtained from three independent experiments. *p , 0.001 versus noninfectedor Ad- b Gal-infected cells.

sion of the transgene was stable over the time period studiedbecause the amounts of BDNF–FLAG mRNA in rats killed 4weeks after the lesion were similar to those in two rats killedat the time of the QUIN lesion (data not shown). TheBDNF–FLAG transcript was not detected on the contralateralside or in the striatum of Ad- b Gal-treated rats.

To determ ine if recombinant BDNF expression, evidencedby anti-FLAG in situ hybridization, corresponds to an overex-pression of BDNF in the striatum, the level of this protein wasmeasured in vivo . Rats were injected in the striatum with eitherAd-BDNF (n 5 5) or Ad- b Gal (n 5 3) or vehicle (n 5 2). Twoweeks later, animals were sacrificed, striata were dissected, and

BDNF content of striatal lysates was quantified using anELISA. The level of BDNF increased from 6 6 1.3 pg ofBDNF/mg of protein in vehicle- and Ad- b Gal-injected striatato 16.6 6 2.7 pg of BDNF/mg of protein in Ad-BDNF-injectedstriata (p , 0.01).

BDNF gene transfer reduces QUIN lesion size

The lesion induced by QUIN injection was examined onDARPP-32-immunostain ed sections. DARPP-32 (dopamineand cyclic AMP-regulated phosphoprotein of 32 kDa) is amarker for the soma and axons of striatal GABA-ergic output


FIG. 3. b -Galactosidase (A, C, and E) and recombinant BDNF (B, D, and F) levels 6 weeks after a single intrastriatal injec-tion of Ad- b Gal or Ad-BDNF, respectively. b -Galactosidase activity was detected enzymatically with 5-bromo-4-chlor o-3-in-dolyl- b -D-galactoside as a substrate, resulting in a blue color. Recombinant BDNF mRNA was detected by anti-FLAG in situhybridization (see Materials and Methods). Transgene expression was detected throughout the caudate putamen (A and B) andthe corpus callosum (E and F). (C and D) Higher magnification of b -galactosidase- and recombinant BDNF-containing cells, re-spectively. Owing to the nuclear-localizing sequence, only the nucleus of b -galactosidase-co ntaining cells is stained, and thesecells therefore appear much smaller than the cells containing recombinant BDNF. Cx, Cortex; cc, corpus callosum; Str, striatum .Scale bar: (A, B, E, and F) 200 m m; (C and D) 50 m m.

neurons (Ouimet et al., 1984). The QUIN-induced lesion wasdefined as the area devoid of immunoreacti vity. Four weeks af-ter QUIN injection, the rats were killed, cryostat serial sectionsencompassing the whole striatum were prepared, and one se-ries of sections was processed for the immunohistoc hemical de-tection of DARPP-32. Lesions appeared smaller in the Ad-BDNF-treated group than in the Ad- b Gal- and sham-treatedgroups (Fig. 4). The lesion area was quantified for each rat bycamera lucida drawings of sections taken every 400 m m throughthe neostriatum . Areas were then integrated to obtain striataland lesion volume. This quantification (Fig. 5A) showed that

lesion volume for the Ad-BDNF-treated group (1.69 6 0.34mm3) was 55% smaller than that for the Ad- b Gal-treated group(3.8 6 0.87 mm3) and 75% smaller than that for the sham-treated group (6.24 6 1.76 mm3). The lesions of the Ad-BDNF-treated group were significantly smaller than those of the othertwo groups (p , 0.05), whereas the Ad- b Gal- and sham-treatedgroups were not significantly different (p 5 0.26).

We studied the overall effect of the lesion on the striatumby determining the volume of this structure. Absolute striatalvolume was measured (Fig. 5B) and it was found that the QUINlesion caused a shrinkage of the neostriatum that was more pro-


FIG. 4. Representative photomicrograp hs showing DARPP-32-immunostai ned sections of sham-injected (A), Ad- b Gal-injected(C,E,G), Ad-BDNF-injected (D,F,H), or intact (B) striatum. (A,C,D) The QUIN-induced lesion (indicated by asterisks) wasclearly identified by the absence of DARPP-32 staining. The lesion was smaller in Ad-BDNF-treated animals than in Ad- b Gal-or sham-treated animals. (E) Higher magnification of the boundary between the spared (sp.) and QUIN-lesioned (les.) areas. (F)Higher magnification of the spared part of the neostriatum. (G and H) Higher magnification of the lesioned areas shown in (C)and (D), respectively. Scale bar: (A–D) 1 mm; (E) 200 m m; (F–H) 100 m m.

nounced, although not significantly so (p 5 0.11), in the Ad-b Gal-treated group (20.27 6 0.89 mm3) than in the Ad-BDNF-treated group (22.27 6 0.75 mm3). In the sham-treated group,the shrinkage was intermediate (21.01 6 1.35 mm3; p 5 0.43versus Ad-BDNF treatment; p 5 0.66 versus Ad- b Gal treat-ment). Taken together, these results suggest that in the Ad-BDNF- and Ad- b Gal-treated groups, the neostriatum shrinkagewas due not only to the QUIN lesion, but also partly to the ade-novirus injection, which is associated with a certain degree oftoxicity.

Histological processing may cause differences between ani-mals. Therefore, the relative lesion volume, defined as the per-centage of the total striatum occupied by the lesion, was cal-culated for each rat. The relative lesion volume in theAd-BDNF-treated group was significantly smaller than thoseof the Ad- b Gal-treated group (p , 0.05) and sham-treatedgroup (p , 0.05; Fig. 5C). This confirmed the results obtainedby absolute lesion volum e measurement. The Ad- b Gal- andsham-treated groups were again not statistically different (p 50.21).

Rescue of DARPP-32-positive neurons by BDNF gene transfer

The effect of recom binant BDNF on the QUIN lesion wasexamined at the cellular level by quantifying the survival ofDARPP-32-positive neurons. For each rat, at the rostrocaudal

level where the area of the QUIN lesion was maximal, threesections were selected and DARPP-32-positive neurons werecounted on these sections on the ipsi- and contralateral sides.Cell density and striatal area were calculated for each rat andexpressed as a percentage of the intact side (Fig. 6). As previ-ously observed with camera lucida drawings, the shrinkage ofthe neostriatum was more pronounced in the Ad- b Gal-treatedgroup (87 6 1.5%) than in the Ad-BDNF-treated group (92.5 62%; p 5 0.03 versus Ad- b Gal treatment) whereas shrinkage inthe sham-treated group (92 6 3%; p 5 0.49 versus Ad-BDNFtreatment) was similar to that in the Ad-BDNF-treated group(Fig. 6B). In contrast, cell density measurements showed thatDARPP-32-positive cells were protected only in the Ad-BDNFgroup (69 6 4%) and not in Ad- b Gal (54 6 7%; p 5 0.04 ver-sus Ad-BDNF treatm ent) and sham (55 6 10%; p 5 0.12 ver-sus Ad-BDNF treatment) groups (Fig. 6A). The total cell num-ber was deduced from area and cell density measurement ineach animal. Ad-BDNF was found to have significantly pro-tected the medium spiny projection neurons of the neostriatum .We found that 46% ( 6 5%) of cells survived in Ad b Gal-treatedanimals, whereas 64% ( 6 4%) survived in Ad-BDNF-treatedanimals (Fig. 6C, p 5 0.01). In terms of total cell number, com-parison between sham- and Ad-BDNF-treated animals resultedin a p value of 0.19. This lack of significance could be explainedby the QUIN lesion variability, which appeared much more pro-nounced in the sham-treated animals than in the adenovirus-treated animals.


FIG. 5. Quantification of lesion and striatalvolume based on DARPP-32-immunostain edsections. (A) Absolute lesion volume deducedby integration of lesioned areas determinedevery 400 m m across the neostriatum. (B) To-tal striatal volum e (lesioned plus spared areas)determined on the same sections as for (A). (C)Relative lesion volume, expressed as a per-centage of whole neostriatum volum e. *p ,0.05 versus Ad- b Gal- and sham-treated groups.

DISCUSSION

In this article we have described the effective in vivo trans-fer of the BDNF gene, using a recombinant adenovirus vector.In a rodent excitotoxic model of HD, we demonstrated that theadenovirus-med iated transfer of BDNF protected the medium-sized spiny neurons of the neostriatum, the principal target ofneurodegeneration in HD. This is the first in vivo evidence thatBDNF is of value for HD treatment.

Effect of BDNF gene transfer in vitro

BDNF has been reported to promote the differentiation ofGABA-ergic neurons. In particular, it increases the number ofcalbindin-im munoreactive cells in primary striatal cultures(Mizuno et al., 1994; Ventimiglia et al., 1995). Consistent withthese observations, we found that the infection of primary stri-atal cultures with Ad-BDNF resulted in an increase in the num-ber of calbindin-imm unoreactive neuronal cells (Fig. 2B). Thus,striatal cells produced the protein encoded by the transgene af-ter adenovirus-m ediated gene transfer. This result also demon-strated that the addition of the FLAG peptide to the carboxy-terminal part of the recom binant BDNF did not affect itsbiological activity.

Effect of BDNF gene transfer in an in vivomodel of HD

Several observations suggest that BDNF is an important fac-tor for mature striatal neurons and that it should therefore be

considered as a putative therapeutic agent for HD. EndogenousBDNF and the mRNA encoding its high-affinity receptor TrkBhave been detected in the adult neostriatum (Altar et al., 1994;Conner et al., 1997). The endogenous BDNF is transported tothe cortical nerve terminals by anterograde transport (Altar etal., 1997), and the TrkB receptor is primarily located in thepostsynaptic densities (Wu et al., 1996). Moreover, BDNF hasbeen reported to be implicated in the induction of the DARPP-32 phenotype in the developing striatum (Ivkovic et al., 1997).

We tested the therapeutic value of BDNF for HD in an ex-citotoxic model of the disease obtained by intrastriatal injectionof QUIN. This procedure reproduces the cell loss observed inHD and can be used to evaluate experimental treatments bycomparing QUIN-induced lesions in treated and nontreated an-imals. Thus, this model of HD is highly relevant for testing ex-perimental therapy, and we have shown here that the prior treat-ment by intracerebral injection of an adenovirus expressing theBDNF gene protected the striatal neurons from the QUIN-in-duced lesion. More recently, transgenic models of HD havebeen developed. In these models, despite the reduced life spanand the behavioral abnormalities, there is no clear neuronal celldeath (Price et al., 1998), which makes these models difficultto use in testing a neuroprotective approach. Nevertheless, theycould prove useful in testing the ability of BDNF to prevent theneuronal dysfunction that occurs in these models.

The mechanism by which BDNF protects against excitotoxicinsult is unknow n. However, it has been suggested that the pro-tection results from the action of BDNF on the transcription ofcalcium-binding proteins (Nakao et al., 1995). Studies in vitro


FIG. 6. Rescue of DARPP-32-positive cells byadenovirus-med iated transfer of the BDNF gene.(A) Density of the DARPP-32 cell populationevaluated for each rat from three sections selectedat the rostrocaudal level where QUIN lesion areawas maximal. (B) Evaluation of the striatal sizeon the same three sections. (C) Total number ofcells in the DARPP-32-positive population, de-duced from cell density and striatal area values.Data are expressed as a percentage of the intactside. *p , 0.05 versus Ad- b Gal. In (A) and (B)comparisons were made using the Student t test.

have shown that BDNF increases the expression of calbindin,calretinin, and parvalbumin (Mizuno et al., 1994; Widmer andHefti, 1994; Ventimiglia et al., 1995). Calbindin levels inBDNF knockout mice are also lower than normal (Jones et al.,1994). As excitotoxicity challenged with an NMDA agonist,such as QUIN, is known to increase the intracellular calciumconcentration (reviewed in Choi, 1994), upregulation of the ex-pression of calcium-binding protein levels may rescue striatalcells from excitotoxic cell death by increasing their capacity tobuffer intracellular calcium (Mattson et al., 1991).

The neuroprotective effect of Ad-BDNF treatment reportedin this study could also be explained by the antiapoptotic prop-erty of BDNF. Apoptotic cell death has been implicated in bothHD and the QUIN-induced rat model of HD (Portera-Cailli auet al., 1995; Ona et al., 1999; Sanchez et al., 1999). Althoughthe effect of BDNF on antiapoptotic proteins is not documented,BDNF was shown to counteract apoptosis in several models ofneurodegeneration (Kaal et al., 1997; Bhave et al., 1999). Inparticular, in an in vitro model of HD, the apoptosis observedafter transfection of primary striatal neurons with a polygluta-mine construct was abolished when BDNF was added in theculture medium (Saudou et al., 1998).

Efficiency of adenovirus-mediated transfer of theBDNF gene

The transfer of the BDNF gene in the QUIN model of HDhas previously been reported. However, although neural rescuetended to be greater in BDNF-treated animals, Martinez-Ser-rano and Björklund have shown that the effects were not sig-nificant for several parameters, including lesion size andDARPP-32 neuron survival (Martinez-Serrano and Björklund,1996). In another study, by Frim et al., it has been reported thatexogenous BDNF has no effect on the excitotoxic lesion (Frimet al., 1993). The lack of agreem ent of these two reports withour results may be due to differences in the gene transfer pro-cedure.

In these studies, the BDNF gene was delivered by an ex vivogene transfer method involving retroviral vectors. However, dif-ferent types of transplanted cells were used for the two studies.Frim et al. used transplants of immortalized fibroblast cells,whereas Martinez-Serran o and Björklund used transplants ofconditionally immortalized neural progenitor cells. The twostudies confirm the importance of the local production of BDNFin that the immortalized fibroblasts remained at the injectionsite (i.e., the corpus callosum) and did not protect the neostria-tum (Frim et al., 1993), whereas conditionally immortalizedneural progenitor cells migrated throughout the neostriatum ,giving mild protection (Martinez-Serra no and Björklund, 1996).

In this article, we used a gene transfer procedure consistingof the direct intracerebral injection of a recom binant adenovi-ral vector. This procedure leads to strong expression of thetransgene in a large area around the injection site (Le Gal LaSalle et al., 1993). Thus, in the paradigm of neuroprotection byBDNF in the QUIN lesion model, the in vivo gene transfermethod used here appears to be more efficient than ex vivo genetransfer. This may be because more BDNF is produced in situafter in vivo gene transfer, resulting in better diffusion of thefactor. We and others have also demonstrated that the neuro-protection provided by the adenovirus-m ediated transfer ofgenes encoding neurotrophic factors is not restricted to the

transfected cells, but extends to neighboring cells (Castel-Barthe et al., 1996; Baumgartner and Shine, 1997; Bilang-Bleuel et al., 1997).

Potential of adenoviral vectors for gene transfer

Consistent with previous studies (Bilang-Bleuel et al., 1997),we report here that a first-generation adenoviral vector drovethe in situ production of a therapeutic protein. However, thisvector appeared to have a toxic effect on the cerebralparenchym a after intracerebral injection (Byrnes et al., 1995).In our study, the striatum was slighly, but not significantly,smaller in Ad- b Gal-treated animals than in sham-treated ani-mals (Figs. 5B and 6B). The striatal size measured for Ad-BDNF-treated animals suggests that the recombinant BDNFprotects against both the QUIN lesion and adenoviral toxicity.The reasons for adenoviral toxicity are unclear, but severalmechanisms, including the production of viral proteins and anti-gen-mediated cytotoxicity, are thought to be involved (Byrneset al., 1995, 1996). Use of new-generation adenoviruses, whichare less toxic and less immunogenic (Dedieu et al., 1997), orof lentiviral vectors, which are efficient agents for gene trans-fer in the CNS (Blömer et al., 1997), is likely to increase thetherapeutic value of neurotrophic gene transfer. This type ofvector, which should allow safe and sustained transgene ex-pression, will be of particular value for testing transfer of theBDNF gene in preclinical animal models of HD, such as chronic3-nitropropionic acid intoxication of primates. These modelswill permit the evaluation of potential clinical benefits of BDNFgene transfer in restorative or neuroprotective approaches forHD over the longer term.

ACKNOWLEDGMENTS

We thank Dr. M. Ghee for critical reading of the manuscriptand Drs. Greengard and Hemmings for giving us the DARPP-32 antibody. This work was funded by the European Commu-nity BIOMED2 Program (BMH 4.CT 96-1012), the Centre Na-tional pour la Recherche Scientifique, the Conseil Régionald’Ile-de-France, the Association Française contre les My-opathies, and by Rhône-Poulenc Rorer. A.-P.B. was supportedby the Fondation Marcel Mérieux and the Association Hunt-ington France.

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Address reprint requests to:Dr. Jacques Mallet

LGN-Bât CERVI83 Bd de l’Hôpital

75013 Paris, France

E-mail: mallet@infobi ogen.fr

Received for publication April 28, 1999; accepted after revi-sion September 22, 1999.


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