Lentiviral Vectors as a Gene Delivery System in the Mouse Midbrain: Cellular and Behavioral Improvements in a 6-OHDA Model of Parkinson's Disease Using GDNF

Experimental Neurology 164, 15–24 (2000) doi:10.1006/exnr.2000.7409, available online at http://www.idealibrary.com on

Lentiviral Vectors as a Gene Delivery System in the Mouse Midbrain: Cellular and Behavioral Improvements in a 6-OHDA Model of Parkinson’s Disease Using GDNF Jean-Charles Bensadoun, Nicole De´glon, Jack L. Tseng, Jean-Luc Ridet, Anne D. Zurn, and Patrick Aebischer Division of Surgical Research and Gene Therapy Center, Pavillon 4, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland Received November 14, 1999; accepted January 5, 2000

ministration, and (iii) instability of the molecules. Efforts are thus being made to optimize the delivery of therapeutic molecules close to the injured neurons. Rodent models of neurodegenerative diseases provide appropriate systems to evaluate new delivery techniques in the central nervous system. Transgenic mice expressing human disease-associated mutations such as transgenic models of amyotrophic lateral sclerosis (13), Huntington’s disease (14, 27), and Alzheimer’s disease (11) represent the most relevant models of the human condition. The recent discovery of genetic mutations in the ␣-synuclein gene in parkinsonian families and deletion in the parkin gene of autosomal recessive juvenile parkinsonism (19, 32) may also lead to the development of transgenic mouse models of Parkinson’s disease (PD). We therefore developed an in vivo gene delivery technique based on a viral vector to screen potential therapeutic molecules in mouse models of neurodegenerative diseases. Replication-defective lentiviral vectors derived from HIV-1 have recently been used as a gene delivery system because they (i) can infect both dividing and nondividing cells in vitro and in vivo, (ii) do not encode viral proteins that may elicit an immune response, and (iii) have a cloning capacity of approximately 9 Kb (9, 31, 39, 40). Sustained expression of the transgene for up to 6 months has been observed in various neuronal populations after injection of LacZ-expressing lentiviral particles in the rat brain (5, 30). To further improve vector biosafety, the four accessory genes of HIV (vif, vpr, vpu, and nef), as well as the U3 region of the 3⬘ long terminal repeat (giving rise to self-inactivating (SIN) vectors), have been deleted (9, 18, 29, 39, 40). Furthermore, the genome has been split into four plasmids to limit the formation of replication-competent particles (9). Finally, to increase transgene expression, the posttranscriptional cis-acting regulatory element of the woodchuck hepatitis virus (WHV) has been inserted (38).

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson’s disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson’s disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson’s disease. © 2000 Academic Press Key Words: lentivirus; mouse; midbrain; Parkinson’s disease; GDNF; animal model.

INTRODUCTION

Delivery of therapeutic molecules to the central nervous system represents an important challenge for the treatment of neurodegenerative diseases. Limitations to overcome include (i) the presence of the blood– brain barrier, (ii) side effects associated with systemic ad15

0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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PD is a neurodegenerative disorder characterized by resting tremor, bradykinesia, rigidity, and postural reflex impairment. While several populations of neurons are affected by the disease, these clinical symptoms are associated with the massive depletion of dopamine resulting from the degeneration of dopaminergic neurons in the substantia nigra (SN). Although conferring initial benefits, systemic L-DOPA treatment leads with time to side effects such as motor fluctuations and dyskinesia (28). Beyond symptomatic approaches, efforts are being made to develop neuroprotective strategies which could slow down or halt disease progression. The recent discovery of potent neuroprotective molecules for dopaminergic neurons represents a promising perspective for the treatment of PD. For instance, glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-␤ superfamily (25), efficiently protects dopaminergic neurons against cell death in various animal models of PD such as 6-hydroxydopamine (6-OHDA)and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced toxicity (12, 17, 34), as well as medial forebrain bundle axotomy (2, 35). In order to assess the efficacy of lentiviral vectors as a gene delivery system in the mouse midbrain, we have first used a vector encoding the reporter gene LacZ to evaluate the type of cells infected and the diffusion of the viral particles. We report that lentiviral vectors can infect a large number of cells, including dopaminergic neurons and astrocytes, and diffuse over long distances when injected close to the substantia nigra of C57BL/6j mice. In a second step, we have assessed the ability of the vector encoding the GDNF gene to protect nigral dopaminergic neurons against 6-OHDA-induced toxicity. We demonstrate that localized expression of GDNF is able to protect nigral dopaminergic neurons against toxin-induced cell death and to reverse apomorphineinduced rotation in the absence of deleterious side effects. MATERIALS AND METHODS

Animals Male C57BL/6j mice (8 weeks old; IFFA CREDO, France) were used in this study. The animals were housed under controlled temperature and humidity with a standardized light/dark cycle (12/12 h) and had free access to food and water. The experiments were carried out in accordance with the European Community Council Directive (86/609/EEC) for care and use of laboratory animals. Lentiviral Vector Production The generation of viral particles was performed with a four-plasmid system. The cDNA coding for a nuclearlocalized ␤-galactosidase (LacZ), the human GDNF containing a Kozak consensus sequence (a 636-bp frag-

ment: positions 1–151 and 1– 485; GenBank Accession Nos. L19062 and L19063), or a mutated GDNF (deletion of amino acids 74 – 85 of the mature GDNF, as described by Choi-Lundberg and colleagues (7)) were cloned in the SIN-W-PGK transfer vector (10). The packaging construct used in this study was the pCMV⌬R-8.92 (derived from the pCMV⌬R-8.91 plasmid: destruction of the BamHI restriction site in the coding region of the rev gene). To further decrease the risk of recombination and production of replicationcompetent retroviruses, the Rev gene was inserted in a pRSV-Rev plasmid. The viral particles were pseudotyped with the vesicular stomatitis virus G protein encoded by the pMD.G plasmid as described previously (9, 31). The viral particles were produced by transient transfection of 293T cells (31). Forty-eight hours later, the supernatant was collected and filtered, and the particle content was determined by p24 antigen ELISA assay (Nen™ Life Science, Boston, MA). High-titer stocks were obtained by ultracentrifugation. The pellet was resuspended in phosphate-buffered saline containing 1% bovine serum albumin and stored frozen at ⫺80°C. The batches of virus were tested for the absence of replication-competent viral vectors as previously described (31). The titers of the LacZ stocks were determined on 293T cells. The cells were plated at a density of 2 ⫻ 10 5 cells per well on six-well tissue culture dishes (Costar). Serial dilutions of the viral stocks were added and the number of LacZ-infected cells was analyzed 48 h later. Titers were calculated by counting the number of blue foci per well and dividing it by the dilution factor. LacZ-expressing viruses containing 3–7 ⫻ 10 8 TU/ml were usually obtained. LacZ- (lentiLacZ), GDNF- (lenti-GDNF), or mutated GDNF- (lentimuGDNF) expressing viruses matched for particle content (150,000 ng p24 antigen/ml as measured by ELISA assay) were used for in vivo experiments. The experiments were performed in biosafety level 2 laboratories. Injection of the Lentiviral Vector Stereotaxic injections were performed under pentobarbital anesthesia (60 mg/kg ip) using a 10-␮l Hamilton syringe with a 33-gauge blunt tip needle. The injection was performed just above the SN (anterior ⫺2.9, lateral 1.3, ventral 4.2) as calculated from bregma and the skull surface. The skin was opened and the skull was delicately bored with a 0.5-ml insulin syringe equipped with a 30-gauge needle (Becton Dickinson Europe, France). C57BL/6j mice were unilaterally injected above the right SN with 1 ␮l of concentrated lentiviral stock slowly infused at the speed of approximately 0.2 ␮l/min. Four mice were injected with the lenti-LacZ virus and sacrificed 2 weeks later in order to evaluate the diffusion of the viral particles and the type of infected cells. To investigate the ability of a lentiviral vector

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encoding the GDNF gene to protect nigral dopaminergic neurons against 6-OHDA-induced toxicity, mice were injected with either lenti-GDNF (n ⫽ 7) or lentimuGDNF (n ⫽ 8) and allowed to recover for 2 weeks prior to striatal 6-OHDA injection. For characterization of ␤-galactosidase expression, the mice were sacrificed with an overdose of pentobarbital and perfused transcardially with phosphate-buffered saline containing 0.02% ascorbic acid and with 4% paraformaldehyde. The brains were postfixed in a solution of 4% paraformaldehyde for 24 h and then placed in 25% sucrose for an additional 24 h. The brains were frozen on dry ice and 20-␮m coronal sections were cryocut throughout the entire SN and stored at 4°C in phosphate-buffered saline.

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ical detector (Millipore, Bedford, MA) as previously described (3). In order to normalize the amount of DA in each striatal extract, protein content was analyzed using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Striatal DA content was expressed as picomoles of DA per microgram protein. Tyrosine Hydroxylase (TH) Immunohistochemistry

Animals infected with lenti-GDNF or lenti-muGDNF were injected with 6-OHDA (Aldrich Chemicals, Switzerland) into the right midstriatum (anterior 0.4, lateral 1.8, ventral 3.5) using the same surgical procedure as for the virus injection. Each mouse received 4 ␮g of 6-OHDA dissolved in 2 ␮l of physiological saline containing 0.02% ascorbic acid. The solution was slowly infused at the speed of approximately 0.5 ␮l/min. Following drug treatment, animals were allowed to recover for 2 weeks before behavioral testing.

After removal of a striatal punch, the rest of the brain was postfixed in 4% paraformaldehyde for 24 h and was placed in 25% sucrose for 24 h. Twenty-micrometer coronal sections were cryocut throughout the entire SN and stored at 4°C in phosphate-buffered saline. Free-floating sections were processed for TH immunohistochemistry as previously described (3). Briefly, sections were incubated overnight at 4°C with a monoclonal anti-TH antibody diluted 1:50 (Boehringer Mannheim, Germany), followed by a 2-h incubation at 4°C in biotinylated secondary goat anti-mouse IgG diluted 1:200 (Vector Laboratories, Burlingame, CA). The sections were then incubated in avidin– biotin–peroxidase (Vector Laboratories) diluted 1:100 in phosphate-buffered saline for 2 h at 4°C and revealed with 3,3⬘-diaminobenzidine (Sigma, St. Louis, MO). Sections were mounted onto gelatinized glass slides, dehydrated, cleared in toluene, and coverslipped with Merckoglas (Merck, Germany).

Apomorphine-Induced Rotation

Cell Counting

Apomorphine-induced rotational behavior was assessed at 2, 3, and 4 weeks following 6-OHDA injection. Mice were installed in individual plastic hemispherical bowls (24 cm diameter) and attached to an adapted harness connected to an automated rotometer system (Omnitech Electronics Inc., Columbus, OH). They were allowed to habituate for 10 min before being injected with a subcutaneous dose of apomorphine (0.6 mg/kg; Sigma, St. Louis, MO). Rotational behavior was monitored for 30 min in a closed room to avoid any environmental disturbance. Results were expressed as net turns (ipsilateral– contralateral) per 30 min.

The number of TH-positive cells in the SN was determined by counting every fifth 20-␮m section at a magnification of 100⫻ as previously described (3). Briefly, delimitation between the ventral tegmental area and the substantia nigra was determined by using the medial terminal nucleus of the accessory optic tract as a landmark (24). Estimation of the total number of TH-positive neurons per brain was obtained by multiplying the counts by 5. This approach is a specific case of the method described previously by Abercrombie (1), in that the average neuronal diameter is very similar to the thickness of the sections. The unlesioned side was used as a control for the percentage of remaining neurons (ratio R/L) in each group.

Striatal 6-OHDA Treatment

Determination of Dopamine Content Animals were sacrificed with an overdose of pentobarbital following the last behavioral testing and perfused transcardially with ice-cold phosphate-buffered saline containing 0.02% ascorbic acid. The brain was removed and striata were carefully dissected out using a fresh tissue slicer. A 1.75-mm diameter punch was taken and rapidly frozen on dry ice. Frozen striata (ipsi- and contralateral sides) were weighed, immediately placed in 150 ␮l of a 0.1 N perchloric acid solution and stored at ⫺80°C until analysis. Striatal levels of dopamine (DA) were determined by high-performance liquid chromatography (HPLC) using an electrochem-

Characterization of Infected Cells Immunofluorescence detection using antibodies directed against ␤-galactosidase, the glial marker glial fibrillary acidic protein (GFAP), the marker for neuronal nuclei (NeuN), and TH was performed to determine the proportion of cells double-labeled with LacZ/GFAP, LacZ/NeuN, and LacZ/TH. Free-floating sections (20 ␮m) were first incubated in a blocking solution containing 10% normal goat serum and 0.1% of Triton X-100 in phosphate-buffered saline overnight at 4°C. Double labeling procedures were performed using the following

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FIG. 1. In vivo transduction of LacZ in the region of the substantia nigra (SN) using a lentiviral vector. Sections were stained for the presence of ␤-galactosidase (green) and TH (red). (A) Photomicrograph showing ␤-gal-expressing cells in the SN region. (B) Higher magnification of (A) showing TH-positive cells expressing the ␤-gal reporter protein (nuclear localization). (A) Bar, 100 ␮m; (B) Bar, 20 ␮m.

primary antibodies: rabbit polyclonal anti-␤-galactosidase, 1:500 (5 Prime 3 3 Prime Inc., Boulder, CO); mouse monoclonal anti-GFAP, 1:50 (Boehringer Mannheim, Germany); mouse monoclonal anti-NeuN, 1:200 (Chemicon Int., Temecula, CA); and mouse monoclonal anti-TH, 1:50 (Boehringer Mannheim). Primary antibodies were diluted in the blocking solution and sections were incubated overnight at 4°C. We then incubated the sections in phosphate-buffered saline with the following secondary antibodies for 2 h at room temperature: goat anti-mouse CY3-conjugated antibody, 1:400; and goat anti-rabbit FITC-conjugated antibody, 1:100 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Finally, sections were mounted onto glass slides and coverslipped with Fluorsave Re-

agent (Calbiochem, La Jolla, CA). Counting of doublelabeled cells was performed as described above for the number of TH-positive neurons. hGDNF ELISA Assay Measurement of GDNF expression in the SN was performed using an ELISA assay (R&D Systems, Abingdon, UK). Eight C57BL/6j mice were unilaterally injected close to the right SN with either lenti-GDNF (n ⫽ 4) or lenti-muGDNF (n ⫽ 4) and sacrificed 6 weeks later. Fresh tissue punches of the entire SN region (1.75 ⫻ 2 mm) were rapidly collected and sonicated in phosphate-buffered saline containing 0.5% Triton X-100 and a solution of protease inhibitors (pronase,

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FIG. 2. Identification of LacZ-expressing cells using the neuronal marker NeuN and the astrocytic marker GFAP. (A) Section stained for ␤-galactosidase (green) and NeuN (red). Double-labeled cells are characterized by yellow nuclei (arrowheads). (B) Section stained for ␤-galactosidase (green) and GFAP (red). Arrowheads, astrocytes expressing ␤-gal. Bar, 40 ␮m.

thermolysin, chymotrypsin, trypsin, papain) (Boehringer Mannheim). The samples were centrifuged at 10,000 rpm for 10 min and supernatants were recovered to be processed using an ELISA assay (Promega, Madison, WI). A standard curve was established by adding increasing amounts of recombinant human GDNF to control brain tissue in order to evaluate the percentage of recovery of the extraction procedure and to normalize the results.

Scheffe’s PLSD post hoc test (StatView 4.0). To determine significance, all comparisons were made between the lenti-GDNF and lenti-muGDNF groups. The significance level was set at P ⬍ 0.05. Degree of freedom (df) and F value were also included.

Statistical Analysis

Injection of the LacZ-expressing lentiviral vector above the SN led to ␤-galactosidase expression in 37,966 ⫾ 3400 cells distributed over the entire SN pars compacta (Fig. 1A). The transgene was expressed as

Data were expressed as the mean ⫾ SEM and evaluated for analysis of variance (ANOVA) followed by a

RESULTS

Characterization of Lentiviral Infection in the SN

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FIG. 3. Effect of GDNF on TH-positive nigral cells in 6-OHDA-lesioned mice. Micrographs showing TH immunostaining in animals injected with either lenti-muGDNF (control) (A–C) or lenti-GDNF (D–F). Note the preservation of the number of TH-positive cells as well as the dopaminergic dendritic network in the lenti-GDNF group (F) compared to the control group (C). Arrow, site of needle tract. Bar (A and D), 400 ␮m; bar (B, C, E, and F), 100 ␮m.

far as 1 mm from the injection site, both laterally and rostrocaudally. Double labeling showed that 43.6 ⫾ 4.9% of the LacZ-positive cells stained for the neuronal marker NeuN (Fig. 2A) and 16.5 ⫾ 1.2% for the astrocytic marker GFAP (Fig. 2B). Finally, 20.3 ⫾ 1.7% of the TH-positive nigral dopaminergic neurons expressed the transgene, with a maximum of 40 –50%

␤-gal-expressing dopaminergic neurons close to the needle tract (Fig. 1). Viral stocks used in this study contained between 100,000 and 200,000 ng p24 antigen/ml and were shown to elicit minor tissue reaction characterized by light perivascular cuffing at the level of the needle tract on Nissl-stained sections (data not shown).

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nigral dopaminergic neurons were also visible on cresyl violet (Nissl)-stained sections (Fig. 5). Fresh tissue punches of the SN analyzed by ELISA assay contained 1.95 ⫾ 0.03 ng of human GDNF per punch in lenti-GDNF-injected animals. No GDNF could be detected in the muGDNF-injected mice. Effect of GDNF on Striatal DA Content

FIG. 4. Effect of GDNF on the number of TH-positive nigral cells in 6-OHDA-lesioned animals (ratio lesioned/nonlesioned side). THpositive dopaminergic neurons were significantly protected against 6-OHDA-induced toxicity in mice injected with lenti-GDNF (n ⫽ 7) as compared to lenti-muGDNF-injected animals (n ⫽ 8) (P ⬍ 0.0001). R, right. L, left.

To evaluate the extent of the 6-OHDA lesion and potential protective effects on dopaminergic nerve terminals, the striatal DA content was evaluated using HPLC. Four weeks after 6-OHDA injection, DA was decreased by approximately 90% in both the lentiGDNF (6.75 ⫾ 1.64 pmol DA/␮g protein) and lentimuGDNF-injected animals (4.55 ⫾ 0.87 pmol DA/␮g protein) compared to the unlesioned side (51.6 ⫾ 4.3 and 53.1 ⫾ 4 pmol DA/␮g protein, respectively). The small difference between both groups is not significant (P ⬎ 0.2, ANOVA). Effect of GDNF on Apomorphine-Induced Rotation

Effect of GDNF on the Number of TH-Positive Nigral Cells Immunohistochemical analysis of the SN revealed that GDNF significantly protected TH-positive neurons against 6-OHDA-induced toxicity, with 69.5 ⫾ 5.1% TH-positive neurons on the lesioned side compared to 33.1 ⫾ 4.5% in the muGDNF control group (P ⬍ 0.0001, ANOVA) (Figs. 3 and 4). Moreover, the TH-positive dendritic network was clearly preserved in the GDNF group compared to muGDNF-injected animals (Figs. 3C and 3F). Protective effects of GDNF on

In order to assess a possible functional benefit of GDNF, drug-induced rotation was tested 4, 5, and 6 weeks after viral injection above the SN, i.e., 2, 3, and 4 weeks following injection of 6-OHDA. The injection of apomorphine induced 90 –110 contralateral turns/30 min in muGDNF-injected control mice (Fig. 6). GDNFinjected animals showed less contralateral rotation than the control group during the whole experimental period, reaching a 50% decrease 4 weeks after 6-OHDA injection (P ⬍ 0.05, df ⫽ 1, F ⫽ 5.184; ANOVA). The number of rotations increased with time in the lenti-

FIG. 5. Micrographs showing Nissl-stained section through the SN. Animals injected with lenti-muGDNF (A and B) or lenti-GDNF (C and D). (A and C) Nonlesioned side; (B and D) lesioned side. Bar, 100 ␮m.

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the number of infected nigral dopaminergic neurons, one might consider the injection of a larger volume of viral stock or multiple injection sites. Diffusion analysis of lentiviral particles reveals that ␤-galactosidase staining in the mouse midbrain is found as far as 1 mm from the injection site, both laterally and rostrocaudally. Since the nigral dopaminergic neurons extend over a distance of approximately 1.5 mm, a single 1-␮l injection of concentrated lentiviral stock containing approximately 3 ⫻ 10 8 TU/ml is thus sufficient to express the transgene over the entire SN region. Neuroprotective Effects of GDNF FIG. 6. Effect of GDNF on apomorphine-induced rotation. Animals injected with lenti-muGDNF (n ⫽ 8) or lenti-GDNF (n ⫽ 7) were tested for apomorphine-induced rotation 2, 3, and 4 weeks post-6-OHDA injection. Rotations were expressed as net turns (ipsilateral– contralateral) per 30 min. P ⬍ 0.07, df ⫽ 1, F ⫽ 4.197 at 2 weeks; P ⬎ 0.3, df ⫽ 1, F ⫽ 0.864 at 3 weeks; and *P ⬍ 0.05, df ⫽ 1, F ⫽ 5.184 at 4 weeks post-6-OHDA injection.

muGDNF group, while they remained relatively stable in the lenti-GDNF group during the entire testing period. DISCUSSION

The purpose of the present study was to evaluate the capacity and efficiency of lentiviral vectors to infect the murine midbrain. In addition, a functional study using a GDNF-expressing lentiviral vector was conducted in a murine model of Parkinson’s disease to further validate this delivery system. The rational of this study is driven by the current development of transgenic and knockout mice as models of neurodegenerative diseases and the opportunity to use these models for the screening of biotherapeutic molecules. LacZ Expression in the Substantia Nigra Nigral injection of a lentiviral vector encoding the reporter gene LacZ leads to the expression of the transgene in approximately 40,000 neural cells throughout the entire substantia nigra region. This transduction efficacy is in agreement with that observed after intranigral injection of a LacZ-expressing lentiviral vector in rhesus monkeys in which approximately 200,000 cells were transduced following the injection of five times the volume of concentrated lentivirus (20). In the present study, approximately 45% of the transduced murine cells are NeuN- and 17% GFAP-positive, demonstrating that both neurons and astrocytes can be transduced. An average of 20% of the nigral TH-positive neurons express ␤-gal with a maximum of 40 –50% of the cells close to the needle tract. To further increase

Based on the transduction level and the significant diffusion of lenti-LacZ particles in the murine midbrain, the efficacy of a lentiviral vector encoding the GDNF gene was assessed in a murine model of PD. Our study shows that expression of approximately 2 ng of GDNF in the nigral area prevents the loss of THpositive cells induced by a striatal 6-OHDA lesion. Comparable amounts of GDNF and similar protection levels have been described with GDNF-expressing adenoviral or AAV vectors injected either in the rat striatum or SN (4, 7, 8, 26). Experiments conducted with single or daily bolus injections of microgram quantities of recombinant GDNF close to the SN also led to a similar protection of TH-positive cells (17, 33, 36). However, side-effects such as weight loss were often associated with these large doses (22, 23). Sustained delivery of nanogram quantities of GDNF using a lentiviral vector thus results in comparable neuroprotection levels, while avoiding weight loss. Lentiviral-mediated expression of GDNF in the murine midbrain decreases the apomorphine-induced rotational asymmetry associated with the injection of 6-OHDA by approximately 50%. In comparison, nigral injection of a GDNF-expressing adenoviral vector in rats elicited a 40% decrease in drug-induced turns in one study (21), but other studies using adenoviral or AAV vectors failed to demonstrate any recovery (7, 26). Intranigral doses of hundreds of nanograms of recombinant GDNF had been necessary to obtain similar results in these models (15, 16, 23). This stresses again the advantage of sustained localized expression of low amounts of protein. The reversal of drug-induced rotation observed in our study seems to occur through a mechanism not mediated by striatal dopamine, since the decrease in dopamine associated with the 6-OHDA lesion is not prevented by nigral injection of lentiGDNF. Several studies have previously described such recovery with nigral delivery of GDNF in various models of PD and have hypothesized the involvement of the dopaminergic dendritic network of the SN (6, 15, 21, 23, 35).

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Partial unilateral 6-OHDA lesion of the striatum in mice represents a reproducible and predictable model of PD. It also offers the possibility to assess functional recovery through drug-induced rotational behavior, as long as apomorphine and not amphetamine is used. The choice of apomorphine was driven by its efficacy and reproducibility in the present mouse model, as previously described (37), whereas it was not the case with amphetamine (data not shown). The present study demonstrates that lentiviral vectors can infect a significant number of cells including neurons and astrocytes and diffuse over long distances in the mouse midbrain. The number of cells infected by the GDNF-expressing lentiviral vector and the level of expression of the transgene are sufficient to provide neuroprotection and reverse behavioral deficits in a neurotoxic murine model of PD. Lentiviral vectors therefore should represent a powerful tool for the screening of therapeutic molecules in transgenic and knockout mouse models of neurodegenerative disorders.

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ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Fabienne Pidoux, Maria Rey, and Laurence Winkel. This work was supported by the Swiss National Science Foundation.

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REFERENCES

14.

1. 2.

3.

4.

5.

6.

7.

8.

Abercrombie, M. 1946. Estimation of nuclear population from microtome sections. Anat. Rec. 94: 239 –247. Beck, K. D., J. Valverde, T. Alexi, K. Poulsen, B. Moffat, R. A. Vandlen, A. Rosenthal, and F. Hefti. 1995. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 373: 339 –341. Bensadoun, J. C., O. Mirochnitchenko, M. Inouye, P. Aebischer, and A. D. Zurn. 1998. Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice. Eur. J. Neurosci. 10: 3231–3236. Bilang-Bleuel, A., F. Revah, P. Colin, I. Locquet, J. J. Robert, J. Mallet, and P. Horellou. 1997. Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease. Proc. Natl. Acad. Sci. USA 94: 8818 – 8823. Blo¨mer, U., L. Naldini, T. Kafri, D. Trono, I. M. Verma, and F. H. Gage. 1997. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71: 6641– 6649. Bowenkamp, K. E., A. F. Hoffman, G. A. Gerhardt, M. A. Henry, P. T. Biddle, B. J. Hoffer, and A. C. Granholm. 1995. Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons. J. Comp. Neurol. 355: 479 – 489. Choi-Lundberg, D. L., Q. Lin, Y. N. Chang, Y. L. Chiang, C. M. Hay, H. Mohajeri, B. L. Davidson, and M. C. Bohn. 1997. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275: 838 – 841. Choi-Lundberg, D. L., Q. Lin, T. Schallert, D. Crippens, B. L. Davidson, Y. N. Chang, Y. W. L. Chiang, J. A. Qian, L. Bardwaj, and M. C. Bohn. 1998. Behavioral and cellular protection of rat

15.

16.

17.

18.

19.

20.

21.

23

dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor. Exp. Neurol. 154: 261– 275. Dull, T., R. Zufferey, M. Kelly, R. J. Mandel, M. Nguyen, D. Trono, and L. Naldini. 1998. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72: 8463– 8471. De´glon, N., J. L. Tseng, J. C. Bensadoun, A. D. Zurn, Y. Arsenijevic, L. Pereira de almeida, R. Zufferey, D. Trono, and P. Aebischer. 2000. Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson’s disease. Hum. Gene Ther. 11: 179 –190. Games, D., D. Adams, R. Alessandrini, R. Barbour, P. Berthelette, C. Blackwell, T. Carr, J. Clemens, T. Donaldson, F. Gillespie, T. Guido, S. Hagopian, K. Johnson-Wood, K. Khan, M. Lee, P. Leibowitz, I. Lieberburg, S. Little, E. Masliah, L. Mcconlogue, M. Montoya-Zavala, L. Mucke, L. Paganini, E. Penniman, M. Power, D. Schenk, P. Seubert, B. Snyder, F. Soriano, H. Tan, J. Vitale, S. Wadsworth, B. Wolozin, and J. Zhao. 1995. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature 373: 523–527. Gash, D. M., Z. Zhang, A. Ovadia, W. A. Cass, A. Yi, L. R. Simmerman, D. Russell, D. Martin, P. A. Lapchak, F. Collins, B. J. Hoffer, and G. A. Gerhardt. 1996. Functional recovery in parkinsonian monkeys treated with GDNF. Nature 380: 252– 255. Gurney, M. E., H. Pu, A. Y. Chiu, M. C. Dal canto, C. Y. Polchow, D. D. Alexander, J. Caliendo, A. Hentati, H-X. Deng, W. Chen, P. Zhai, R. L. Sufit, and T. Siddique. 1994. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264: 1772–1775. Hodgson, J. G., N. Agopyan, C. A. Gutekunst, B. R. Leavitt, F. Lepiane, R. Singaraja, D. J. Smith, N. Bissada, K. Mccutcheon, J. Nasir, L. Jamot, X. J. Li, M. E. Stevens, E. Rosemond, J. C. Roder, A. G. Phillips, E. M. Rubin, S. M. Hersch, and M. R. Hayden. 1999. A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23: 181–192. Hoffer, B. J., A. Hoffman, K. Bowenkamp, P. Huettl, J. Hudson, D. Martin, L. F. Lin, and G. A. Gerhardt. 1994. Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo. Neurosci. Lett. 182: 107–111. Hoffman, A. F., C. G. Van Horne, S. Eken, B. J. Hoffer, and G. A. Gerhardt. 1997. In vivo microdialysis studies on somatodendritic dopamine release in the rat substantia nigra: effects of unilateral 6-OHDA lesions and GDNF. Exp. Neurol. 147: 130 –141. Kearns, C. M., and D. M. Gash. 1995. GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo. Brain Res. 672: 104 –111. Kim, V. N., K. Mitrophanous, S. M. Kingsman, and A. J. Kingsman. 1998. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J. Virol. 72: 811– 816. Kitada, T., S. Asakawa, N. Hattori, H. Matsumine, Y. Yamamura, S. Minoshima, M. Yokochi, Y. Mizuno, and N. Shimizu. 1998. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392: 605– 608. Kordower, J. H., J. Bloch, S. Ma, Y. Chu, S. Palfi, B. Roitberg, M. Emborg, P. Hantraye, N. De´glon, and P. Aebischer. 1999. Lentiviral gene transfer to the nonhuman primate brain. Exp. Neurol. 160(1): 1–16. Lapchak, P. A., D. M. Araujo, D. C. Hilt, J. Sheng, and S. S. Jiao. 1997. Adenoviral vector-mediated GDNF gene therapy in a rodent lesion model of late stage Parkinson’s disease. Brain Res. 777: 153–160.

24 22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

BENSADOUN ET AL. Lapchak, P. A., S. S. Jiao, P. J. Miller, L. R. Williams, V. Cummins, G. Inouye, C. R. Matheson, and Q. Yan. 1996. Pharmacological characterization of glial cell line-derived neurotrophic factor (GDNF): Implications for GDNF as a therapeutic molecule for treating neurodegenerative diseases. Cell Tissue Res. 286: 179 –189. Lapchak, P. A., P. J. Miller, F. Collins, and S. Jiao. 1997. Glial cell line-derived neurotrophic factor attenuates behavioural deficits and regulates nigrostriatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: Comparison of intraventricular and intranigral delivery. Neuroscience 78: 61–72. Lee, C. S., H. Sauer, and A. Bjo¨rklund. 1996. Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat. Neuroscience 72: 641– 653. Lin, L. F., D. H. Doherty, J. D. Lile, S. Bektesh, and F. Collins. 1993. GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130 –1132. Mandel, R. J., S. K. Spratt, R. O. Snyder, and S. E. Leff. 1997. Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of parkinsons disease in rats. Proc. Natl. Acad. Sci. USA 94: 14083–14088. Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. W. Davies, and G. P. Bates. 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493–506. Marsden, C. D., and J. D. Parkes. 1976. “On-off” effects in patients with Parkinson’s disease on chronic levodopa therapy. Lancet 1: 292–296. Miyoshi, H., U. Blo¨mer, M. Takahashi, F. H. Gage, and I. M. Verma. 1998. Development of a self-inactivating lentivirus vector. J. Virol. 72: 8150 – 8157. Naldini, L., U. Blo¨mer, F. H. Gage, D. Trono, and I. M. Verma. 1996. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93: 11382–11388. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery

32.

33.

34.

35.

36.

37.

38.

39.

40.

and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267. Polymeropoulos, M. H., C. Lavedan, E. Leroy, S. E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E. S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W. G. Johnson, A. M. Lazzarini, R. C. Duvoisin, G. Di Iorio, L. I. Golbe, and R. L. Nussbaum. 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276: 2045–2047. Sauer, H., C. Rosenblad, and A. Bjo¨rklund. 1995. Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion. Proc. Natl. Acad. Sci. USA 92: 8935– 8939. Tomac, A., E. Lindqvist, L-F. H. Lin, S. O. Ogren, D. Young, B. J. Hoffer, and L. Olson. 1995. Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature 373: 335–339. Tseng, J. L., E. E. Baetge, A. D. Zurn, and P. Aebischer. 1997. GDNF reduces drug-induced rotational behavior after medial forebrain bundle transection by a mechanism not involving striatal dopamine. J. Neurosci. 17: 325–333. Winkler, C., H. Sauer, C. S. Lee, and A. Bjo¨rklund. 1996. Short-term GDNF treatment provides long-term rescue of lesioned nigral dopaminergic neurons in a rat model of Parkinson’s disease. J. Neurosci. 16: 7206 –7215. Winkler, J., and B. Weiss. 1986. Reversal of supersensitive apomorphine-induced rotational behavior in mice by continuous exposure to apomorphine. J. Pharmacol. Exp. Ther. 238(1): 242–247. Zufferey, R., J. E. Donello, D. Trono, and T. J. Hope. 1999. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73: 2886 –2892. Zufferey, R., T. Dull, R. J. Mandel, A. Bukovsky, D. Quiroz, L. Naldini, and D. Trono. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72: 9873– 9880. Zufferey, R., D. Nagy, R. J. Mandel, L. Naldini, and D. Trono. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotech. 15: 871– 875.