Local GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in SOD1G93A transgenic mice

www.elsevier.com/locate/ynbdi Neurobiology of Disease 16 (2004) 139 – 149

Local GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in SOD1G93A transgenic mice Sandrine Guillot, a Mimoun Azzouz, b Nicole De´glon, a Anne Zurn, c and Patrick Aebischer a,* a

Institute of Neurosciences, Swiss Federal Institute of Technology Lausanne, EPFL, Lausanne, Switzerland Oxford BioMedica Ltd., Oxford, UK c Department of Experimental Surgery, Lausanne University Medical School, 1015, Lausanne, Switzerland b

Received 27 May 2003; revised 16 December 2003; accepted 15 January 2004 Available online 9 April 2004

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are associated with mutations in the cytosolic Cu/Zn superoxide dismutase 1 (SOD1) gene. Transgenic SOD1 mice constitute useful models of ALS to screen therapeutical approaches. Glial cell line-derived neurotrophic factor (GDNF) holds promises for the treatment of motoneuron disease. In the present study, GDNF expression in motoneurons of SOD1G93A transgenic mice was assessed by facial nucleus or intraspinal injection of lentiviral vectors (LV) encoding GDNF. We show that lentiviral vectors allow the expression for at least 12 weeks of GDNF that was clearly detected in motoneurons. This robust intraspinal expression did, however, not prevent the loss of motoneurons and muscle denervation of transgenic mice. In contrast, LV-GDNF induced a significant rescue of motoneurons in the facial nucleus and prevented motoneuron atrophy. The differential effect of GDNF on facial nucleus versus spinal motoneurons suggests different vulnerability of motoneurons in ALS. D 2004 Elsevier Inc. All rights reserved. Keywords: Glial cell line-derived neurotrophic factor; Lentiviral vector; Amyotrophic lateral sclerosis

Introduction Amyotrophic lateral sclerosis (ALS) is a lethal, paralytic disorder caused by the progressive degeneration of motoneurons in the spinal cord, brainstem and cerebral cortex. Approximately 2% of all ALS cases arise as a dominantly inherited trait named familial ALS and are associated with more than 90 different missense mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase 1 (SOD1) (Rowland and Shneider, 2001). Overexpression of various SOD1 mutations in transgenic mice faithfully reproduces the symptomatology of the disease and constitutes therefore a useful model to * Corresponding author. Institute of Neurosciences, Building SG-AAI, Swiss Federal Institute of Technology Lausanne, EPFL, 1015 Lausanne, Switzerland. Fax: +41-21-693-95-20. E-mail address: [email protected] (P. Aebischer). Available online on ScienceDirect (www.sciencedirect.com.) 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.01.017

screen therapeutical approaches for ALS. Mice carrying a human SOD1 construct with Gly93!Ala (G93A) mutation develop limb weakness, loss of motoneurons, impaired axonal transport, ventral roots Wallerian degeneration and muscle denervation atrophy (Gurney, 1994; Zhang et al., 1997). Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron factor yet identified (Henderson et al., 1994; Oppenheim et al., 1995; Zurn et al., 1994). GDNF prevents motoneuron degeneration in mice and rats following sciatic or facial nerve axotomy (Hottinger et al., 2000; Houenou et al., 1996; Li et al., 1995; Matheson et al., 1997; Munson and McMahon, 1997; Oppenheim et al., 1995; Yan et al., 1995). GDNF also slows down motoneuron degeneration in the pmn mouse model of progressive motoneuropathy (Sagot et al., 1996), as well as programmed motoneuron cell death during development (Oppenheim et al., 1995). In transgenic mice expressing GDNF in muscle, a hyperinnervation by motoneurons has been reported (Nguyen et al., 1998). These actions are mediated through a heterodimer receptor system composed of the c-ret protein and the GDNF-a receptor (Durbec et al., 1996; Treanor et al., 1996; Trupp et al., 1996) which are expressed in adult rodent motoneurons. Expression of these receptors is up-regulated in motoneurons after nerve injury (Burazin and Gundlach, 1998; Glazner et al., 1998). Moreover, motoneurons can bind, internalize and retrogradely transport GDNF from muscle in a receptor-dependent manner (Yan et al., 1995). Taken together, these observations suggest that GDNF holds promises for the treatment of motoneuron diseases. Retroviral vectors based on the human immunodeficiency virus (HIV) infect non-dividing cells including neurons (Naldini et al., 1996). Stable, long-term expression of the reporter gene h-galactosidase (h-Gal) has been achieved in rodent (Hottinger et al., 2000) and primate (Kordower et al., 1999) brain. Lentiviral vector-mediated delivery of GDNF efficiently prevents the loss of dopaminergic nigrostriatal neurons in rodent (Bensadoun et al., 2000; De´glon et al., 2000) and primate models of Parkinson’s disease (Kordower et al., 2000) as well as axotomyinduced facial motoneuron death in adult mice (Hottinger et al., 2000). The viral vector-mediated delivery of GDNF has been studied in SOD1 transgenic mice. Significant delay in disease progression has been reported by intramuscular injection of adeno-

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Fig. 1. h-Gal expression in the mouse spinal cord 5 weeks following lentiviral vector injection. (A) Nissl-stained transverse section showing the absence of lesion on the injected side. h-Gal staining reveals numerous cells expressing the transgene in transverse (B) and longitudinal (C) sections of the spinal cord at the L4 level. Arrow indicates the injection site. Scale bars: B = 150 Am, C = 100 Am.

viral vectors (Acsadi et al., 2002), adeno-associated vectors (Kaspar et al., 2003; Wang et al., 2002) or an ex vivo gene delivery system in which myoblasts were retrovirally transduced with a GDNF gene (Mohajeri et al., 1999). In the present work, the effect of locally expressed GDNF was assessed in SOD1G93A transgenic mice by direct facial nucleus or intraspinal injection of lentiviral vectors encoding GDNF. The present study was designed to evaluate the transduction efficiency of motoneuron, the cell type specificity of transgene expression and the potential therapeutic effect of GDNF applied directly to the facial nucleus or ventral spinal cord in a SOD1 transgenic mice model of ALS.

Materials and methods Production of recombinant lentiviral vectors The cDNA coding for nuclear-localized-galactosidase (LacZ), human GDNF containing a Kosak consensus sequence (a 636-bp fragment, position 1 – 151 and 1 – 485; GenBank accession numbers L19062 and L19063), or mutated GDNF (deletion of amino acids 74 – 85 of the mature GDNF leading to its absence of the secretion) (Choi-Lundberg et al., 1997) were cloned in the SIN-W-PGK transfer vector (De´glon et al., 2000). This vector contains a 400bp deletion (EcoRV – PvuII) in the U3 region of the 3V-LTR to obtain a self-inactivating vector (SIN) reducing the risk of emergence of replication competent recombinants (Zufferey et al., 1998). This plasmid was further modified by the insertion of the post-

transcriptional cis-acting regulatory element of the woodchuck hepatitis virus (WPRE) (a 587-bp fragment: position 1093 – 1684 of the WHV complete genome: GenBank accession number J04514) (Zufferey et al., 1999) as this element significantly increases transgene expression. The mouse PGK promoter was used as an internal promoter. The packaging construct used in this study was the pCMVDR-8.92 (derived from the pCMVDR-8.91 plasmid: destruction of the BamH1 restriction site in the coding region of the rev gene). To further decrease the risk of recombination and production of replication-competent retroviruses, the Rev gene was inserted in the pRSV-Rev plasmid. The viral particles were pseudotyped with the vesicular stomatitis virus G-protein encoded by the pMD.G plasmids described previously (Naldini et al., 1996). The viral particles were produced by transient transfection of 293T cells (Naldini et al., 1996). Forty-eight hours later, the supernatant was collected and filtered and the particle content was determined by an ELISA assay for p24 (NEN, Boston, MA). Hightiter stocks were obtained by ultracentrifugation. The pellet was resuspended in PBS and 1% BSA and stored frozen at 80jC. The batches of virus were tested for the absence of replication-competent viral vectors (Naldini et al., 1996). Serial dilutions of the viral stocks were added and the number of 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. Viruses ranged from 3 to 7  108 transfection units/ml for in vivo experiments; LV-LacZ, LV-GDNF and LV-mGDNF were matched for particle content (150,000, 150,000 and 100,000 ng p24 antigen/ml, respectively, as measured by ELISA).

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Fig. 2. Transduction of neuronal cells with the LacZ-expressing lentiviral vector. (A) Transverse section stained for h-Gal; (B) same section stained for the neuronal marker NeuN; (C) composite image showing transduced neurons (yellow nuclei). Scale bars: 200 Am. Arrows indicate transduced neurons by LVLacZ. (D) Quantification of h-Gal expression in transverse sections of the mouse spinal cord 5 weeks following LV-LacZ injection. The total number of h-Galpositive cells is represented by open circles. Filled circles indicate cells co-expressing h-Gal and the neuronal marker NeuN.

Fig. 3. Retrogradely fluorogold-labeled motoneurons expressing h-Gal. Staining for h-Gal (A) and fluorogold (B) in spinal motoneurons. Arrows indicate motoneurons back-labeled with fluorogold-expressing h-Gal. (C) Percentage of fluorogold-positive motoneurons expressing the reporter gene h-Gal. Scale bar: 300 Am.

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Animals

Surgery

C57BL6 (Iffa Credo, France) mice were used to evaluate the transduction efficiency of control LV-LacZ in the spinal cord. Transgenic mice overexpressing the G93A SOD1 mutation (G1H line) were used to evaluate the effect of GDNF on spinal and facial motoneuron survival (Gurney, 1994). This line was maintained as a hemizygote by breeding G93A males with female littermates (B6SJL/F1 females; Iffa Credo). The offspring were genotyped by PCR amplification of DNA extracted from the tail tissue. The primer sequences were selected as previously described (Rosen et al., 1993). Mice were housed in isolated cages at room temperature in a 12 h light – dark cycle with free access to food and water. Transgenic mice were sacrificed when they were unable to right themselves within 30 s when placed on their sides (end-stage of disease). All experiments were carried out in accordance to the European Community Council Directive (86/609/EEC) for care and use of laboratory animals.

Intraspinal and facial nucleus injections of lentiviral vectors Forty-day-old mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (62.5 mg/kg body weight). For intraspinal injection, animals were then placed in a stereotaxic frame and their spinal cords immobilized using a spinal adaptor (Stoelting Co., IL, USA). The lentiviral vector solution was injected into the lumbar spinal cord at the L4 level following a laminectomy. The intraspinal injection consists in two bilateral sites (each site separated by 2 mm on each side of the spinal cord) separated by 1.5 mm along the spinal cord. For facial nucleus injection, mice were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). A midline skin incision was performed. The skull was then opened with a dental drill at 6.2 mm caudal and 1.2 mm lateral to the bregma to do a unilateral injection. For both types of injection, lentiviral vector solutions (0.5 Al/site for spinal cord and 1 Al/site for facial nucleus; titer: 100,000 ng p24 antigen/ml) were injected at a rate of

Fig. 4. Expression of GDNF in the spinal cord of a wild-type B6SJL mouse 3 months following a unilateral lentiviral vector injection at the L4 level. (A) Micrographs showing the extensive diffusion of GDNF in the ventral horn of the spinal cord. Serial 20-Am-thick sections separated by 240 Am. Scale bar: 500 Am. Higher power shows expression of GDNF in cell body (B) and neurites (C) of morphologically identified motoneurons. Scale bars: 50 Am.

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0.2 Al/min through a 10-Al Hamilton syringe fitted with a 34-gauge needle, down to a depth of 5.6 mm for facial nucleus and 0.75 mm for spinal cord using an infusion pump (Stoelting). Following the injection, the needle was left in place for an additional 5 min before being retrieved. Fluorogold injection in the sciatic nerve To study the transduction efficiency of LV-LacZ in the spinal cord, C57BL6 wild-type mice who received intraspinal lentiviral vector injection 4 weeks before were anesthetized by an intraperitoneal injection of sodium pentobarbital to do a unilateral injection of fluorogold (FG). The homolateral sciatic nerve was exposed at mid-thigh level and was cut 5 mm proximal to the nerve trifurcation. A small cup containing a 2% FG solution in saline was placed on the proximal segment of the transected nerve. One week after application of FG, the animals were perfused transcardially with 4% paraformaldehyde. The lumbar spinal cord was dissected out and histological analysis was performed as described below. The number of FG and h-Gal double-labeled motoneurons were counted 5 weeks after injection of the viral vector. Histological evaluation SOD1G93A mice were sacrificed at 3 months post-injection and C57BL6 wild types at 5 weeks. Mice were perfused transcardially with a freshly prepared 4% paraformaldehyde solution in phos-

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phate-buffered saline (PBS). The spinal cords and brainstems were excised and cryoprotected in 25% sucrose for 2 days. Twentymicrometer sections were subsequently cut on a cryostat and stained either for Nissl or for immunohistochemistry. For h-Gal, NeuN and GFAP immunolabelings, non-specific binding was blocked by a 2-h incubation with 10% goat serum and 0.1 % Triton X-100 in PBS at room temperature before incubation with specific antibodies: (i) polyclonal rabbit anti-h-Gal diluted 1:500 (5Prime3Prime Inc., USA); (ii) monoclonal anti-NeuN antibody diluted 1:50 (Chemicon, Switzerland); (iii) mouse monoclonal anti-GFAP, 1:50 (Boehringer Mannheim, Germany). Secondary antibodies were added for 2 h after washing the sections [h-Gal staining: goat anti-rabbit FITC diluted 1:100 (Jackson ImmunoResearch Laboratories, Inc., USA); NeuN and GFAP labeling: Cy3 goat anti-mouse at a 1:400 dilution (Jackson ImmunoResearch Laboratories)]. To evaluate the number of neurons and astrocytes infected with the LacZ lentiviral constructs, lumbar spinal cord sections extending 1.5 mm in either direction from the needle tract were analysed. The number of h-Gal-positive cells double-labeled with NeuN or GFAP were assessed on every third section. The proportion of infected motoneurons was assessed as the percentage of FG back-labeled cells expressing h-Gal. Ten serial sections at 0, 0.5, 1 and 1.5 mm distance from the injection site per animal were analysed (n = 5 animals). To evaluate the number of motoneurons in the spinal cord of GDNF-injected SOD1G93A transgenic mice, counts of motoneurons with distinct nuclei and

Fig. 5. Expression of GDNF in the spinal cord of a SOD1G93A transgenic mouse 3 months following a bilateral lentiviral vector injection at the L4 level. (A) GDNF immunostaining showing the extensive diffusion of GDNF covering a distance of up to 2.64 mm along the ventral spinal cord. Serial 20-Am-thick sections separated by 240 Am. Scale bar: 500 Am.

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nucleoli were performed in 12 Nissl-stained 20-Am-thick sections, separated by 240 Am where GDNF immunoreactivity was observed in serial sections. To evaluate the number of spinal motoneurons of wild-type or uninjected SOD1G93A transgenic mice, we took 12 Nissl-stained 20-Am-thick sections, separated by 240 Am of the same area of spinal cord corresponding to the analysed area in GDNF-injected SOD1G93A transgenic mice. Data (means F SEM) refer to the total number of motoneurons on 12 sections along 2.640 mm of spinal cord. To evaluate the number of motoneurons in the facial nucleus, sections stained for Nissl were counted on every fifth section on both the injected and uninjected sides, and the number of cells present in each facial nucleus was multiplied by 5. Morphometric analysis of cells in spinal cord and facial nucleus of 12 and 20 sections, respectively, were performed with Analysis software. Total area measurements were carried out and stored in an MS Excel spreadsheet (Microsoft). To evaluate the diffusion of GDNF in the brainstem and spinal cord, immunohistochemistry using an anti-human GDNF antibody was performed. Briefly, 20-Am-thick sections were first quenched for 20 min in 0.1 M sodium periodate, followed by a 1 h blocking in 100 mM Tris/150 mM NaCl (TBS) containing 2% BSA, 3% normal horse serum (NHS) and 0.5% Triton X-100. They were then incubated for 48 h at room temperature in TBS containing biotinylated goat anti-GDNF antibody (R&D Systems, Wiesbaden, Germany) at a 1:250 dilution, 1% BSA, 1% NHS and 0.04% Triton X-100. The sections were then incubated for 1 h at room temperature in the same buffer containing biotinylated horse anti-goat IgG (1:200) (Vector laboratories, Burlingame, USA), 1% NHS and 1% BSA. Antibody binding was revealed according to standard procedures using an avidin – biotin – peroxidase kit (Vector Laboratories) and DAB (Diaminobenzidin, Sigma, Buchs, Schweiz). Electrophysiological recordings Evoked compound muscle action potential (CMAP) amplitudes were evaluated as previously described (Azzouz et al., 1997). Briefly, electrophysiological recordings across the sciatic nerve segment were made using a Keypoint electromyogram apparatus (Dantec, Skovlunde, Denmark). Measurements were made at 38, 65, 72, 86, 101, 122, 128 and 131 days of age. Mice were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital. The sciatic nerve was stimulated at a paraspinal site. Stimulation consisted of singles 0.2 ms, 1 Hz supra maximal pulses through a unipolar needle electrode (reference 13R81; Dantec). The evoked CMAPs were recorded from the medial part of the gastrocnemius muscle with the same electrode. The CMAP amplitude was measured from peak to peak. Spontaneous fibrillation potentials (SFPs) were recorded at the same days as the CMAP amplitudes with a concentric needle electrode (reference 13R05; Dantec) inserted through the skin into several sites of the gastrocnemius muscle. Only SFPs with an amplitude ranging between 20 and 300 AV were taken into account. Traces showing voluntary contractile activity were discarded. Statistical studies Data were expressed as means F SEM. Data were analyzed for statistical significance using an analysis of variance, ANOVA followed by a post hoc Student t test. The significance level was set at P < 0.05.

Fig. 6. Effect of GDNF on the onset of motor deficiency and evoked compound muscle action potential (CMAP) amplitude in the gastrocnemius muscle, and on the motoneuron size of the lumbar spinal cord in SOD1G93A transgenic mice. (A) Graphical representation of the cumulative probability of disease onset in LV-GDNF-treated mice (n = 6) and control groups (n = 5 per group). Onset of disease is defined by the appearance of SFPs in gastrocnemius muscle. (B) Time evolution of evoked compound muscle action potential (CMAP) amplitude in the gastrocnemius muscle after stimulation of the sciatic nerve in FALS transgenic mice injected with LVGDNF (n = 6), LV-LacZ (n = 5) and untreated animals (n = 5). (*) indicates P < 0.05. (C) Size distribution of spinal motoneurons in the untreated (n = 5) and LV-GDNF-injected (n = 6) SOD1G93A transgenic mice, and in untreated WT mice (n = 5). (*) and ($) indicate P < 0.01 and P < 0.05, respectively.

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Results Analysis of the transduction efficiency by a lentiviral vector coding the lacZ reporter gene in the spinal cord of wild-type mice To determine the transduction efficiency of lentiviral vectors, we performed intraspinal injections of lentiviruses expressing the marker protein h-Gal in wild-type C57BL/6 mice. All mice tolerated the lentivirus injections without noticeable complications. Animals continued to behave normally in their cage, indicating the absence of functional deterioration following intraspinal injection

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of lentivirus. Nissl staining revealed that the injections were only associated with a mild degree of inflammation, with no significant cell damage (Fig. 1A). Numerous infected cells were detected in the spinal cord (Fig. 1B). Both histochemistry and immunofluorescence revealed robust reporter gene expression within the ventral spinal cord. Transverse sections of the spinal cord revealed high transduction efficiency and extensive diffusion of the virus (Figs. 1B and 2D). In longitudinal sections, the majority of h-Galpositive cells were found within 1.0 F 0.3 mm rostral and caudal to the site of viral delivery (Figs. 1C and 2D), the number of infected cells being higher close to the injection site (Fig. 2D).

Fig. 7. Expression of GDNF in the brainstem of a SOD1G93A transgenic mouse 3 months following a unilateral lentiviral vector injection. (A) Immunostaining showing extensive diffusion of GDNF in the facial nucleus. Scale bar: 600 Am. Nissl staining photomicrographs in the uninjected facial nucleus of a wild-type B6SJL control mouse (B) and in the uninjected side (C) and in a LV-GDNF-injected contralateral side (D) of facial nucleus of the same SOD1G93A transgenic mouse. Scale bars: 200 Am. (E) Histogram showing the number of motoneurons in the facial nucleus of each group. A statistically significant difference was observed between the LV-GDNF-injected side (n = 6) versus uninjected side (n = 6) and the LV-mGDNF-injected side (n = 4). (*) indicates P < 0.001.

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To identify the phenotype of the infected cells, sections were co-labeled with antibodies for h-Gal (Fig. 2A) and either NeuN (Fig. 2B) or GFAP. Seventy five F 9.2% of the h-Gal-labeled cells were double-labeled with NeuN (Figs. 2C and 2D), while only scarce cells were double-stained with GFAP (data not shown). To assess the percentage of motoneurons expressing the reporter gene, motoneurons were back-labeled with FG 4 weeks following spinal lentiviral injections (Figs. 3A and 3B). Double-labeled cells were detected as far as 1.5 mm from the injection site (Fig. 3C). In sections surrounding the injection site, 32.9 F 4.5% of the FGlabeled motoneurons expressed h-Gal. The percentage of labeled cells decreased with increasing distance from the injection site (Fig. 3C). The actual number of transduced motoneurons is probably underestimated because only a fraction of motoneurons become FG labelled.

did also not prevent the motoneuron atrophy of the remaining spinal motoneurons in SOD1G93A transgenic mice compared to the wild-type control mice (Fig. 6C). Effect of facial nucleus injection of LV-GDNF in SOD1G93A transgenic mice In contrast to the effect of GDNF in the spinal cord, LVGDNF induced a small but significant rescue of motoneurons in the facial nucleus of SOD1G93A transgenic mice (Fig. 7). The rostro-caudal diffusion of GDNF covered the entire facial nucleus in the injected side 3 months post LV-GDNF injection (Fig. 7A). At the time of death, SOD1G93A transgenic mice showed a significant loss of motoneurons in the facial nucleus compared to the B6SJL control mice (Fig. 7E) ( P < 0.0001). No statistical difference was observed between the mutated GDNF-

Transduction efficiency of lentiviral vectors coding GDNF in the ventral spinal cord of wild-type B6SJL mice To determine the transduction efficiency of a lentiviral vector coding for a diffusible neurotrophic factor, intraspinal injections of lentivirus expressing GDNF were performed in wild-type B6SJL mice, the genetic background of SOD1G93A transgenic mice. The long-term expression of GDNF in spinal cord was evaluated by immunohistochemistry. Fig. 4 reveals that 3 months following a unitaleral L4 injection of a lentiviral vector, GDNF diffused over a significant area (Fig. 4A) of ventral spinal neurons. High-power micrographs show GDNF expression in motoneuron-like cells both at the soma (Fig. 4B) and dendrite (Fig. 4C) level. Effect of intraspinal injections of LV-GDNF in SOD1G93A transgenic mice The effect of GDNF expression was evaluated in SOD1G93A transgenic mice. For that purpose, 5-week-old mice were injected bilaterally at two sites separated by 1.5 mm into the lumbar spinal cord with LV-GDNF. Strong expression of GDNF was observed over a distance of 2.4 F 0.480 mm in ventral spinal cord 120 days post-injection (Fig. 5). To determine the effect of GDNF on the onset of motor deficiency, SFPs were measured in the gastrocnemius muscle between 40 and 140 days of age. SFPs are an indication of muscle fiber denervation. The onset of clinical disease, as defined by the first appearance of SFPs in the gastrocnemius muscle, was not significantly delayed in LV-GDNF-treated mice compared to either LV-LacZ or untreated controls (Fig. 6A). To study the neuromuscular function in FALS transgenic mice, CMAP amplitudes in gastrocnemius muscles were measured between 40 and 140 days as an indication of the number of functional neuromuscular units (Fig. 6B). CMAP values in the gastrocnemius muscle were normal and not significantly different between 40 and 80 days in both LV-GDNF-treated and control SOD1G93A animals (Fig. 6B). Thereafter, the CMAP amplitude decreased progressively with time in all groups up to 90 days. With the exception of one time point, the CMAP amplitude was not different in LV-GDNF-treated mice than in control groups. In accordance with these results, local GDNF expression in the lumbar spinal cord did not protect ventral spinal motoneurons from cell death. LV-GDNF group (n = 6) revealed 36 F 2.9 motoneurons, whereas the untreated group (n = 5) exhibited 34 F 2.7 motoneurons in 12 sections separated by 240 Am. GDNF delivery

Fig. 8. Effect of GDNF on the motoneuron size in the brainstem of a SOD1G93A transgenic mouse 3 months following a unilateral lentiviral vector injection. Nissl staining photomicrographs in the uninjected side (A) and in a LV-GDNF-injected contralateral side (B) of facial nucleus of the same SOD1G93A transgenic mouse. Scale bars: 50 Am. Size distribution of facial motoneurons in the uninjected side (n = 6) versus control wild type (n = 4) (C) and in LV-GDNF-injected side (n = 6) versus LV-mGDNF-injected side (n = 4) of facial nucleus (D). (*) and ($) indicate P < 0.01 and P < 0.05, respectively.

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injected and uninjected sides of SOD1G93A transgenic mice ( P > 0.1) (Fig. 7E). Injection of LV-mutated GDNF had no protective effect on the facial nucleus motoneurons (Fig. 7E). In contrast, SOD1G93A transgenic mice injected with LV-GDNF showed a significant rescue of facial motoneurons as compared to the uninjected sides ( P < 0.001) or the sides injected with LVmutated GDNF ( P < 0.01). At 4 months, facial motoneurons of SOD1G93A transgenic mice were characterized by the presence of a cellular atrophy compared to wild-type control mice (Figs. 7B and 7C). Mice injected with the mutated GDNF vector showed the same atrophy as the uninjected side, whereas SOD1 mice injected with the GDNF vector showed a significant preservation of the size distribution profiles in the remaining motoneurons (Figs. 7D and 8).

Discussion In the present study, we demonstrate the ability of a lentiviral vector to express a transgene directly in the ventral spinal cord and facial nucleus of mice over an extended area and for at least 12 weeks. This study also reveals the ability of locally delivered GDNF to allow a partial protection of motoneurons in facial nucleus of SOD1G93A transgenic mice, whereas it does not in the spinal cord. Expression of a transgene in the spinal cord and facial nucleus of mice requires an ad hoc developed system composed of a stereotaxic frame and an automatic micropump allowing the localized injection of hundreds of nanoliters of viral stock solution without inducing any significant damage. Provided these precautions, lentiviral-mediated expression of transgenes leads to a minimal inflammatory response comparable to other CNS injection sites (Bensadoun et al., 2000; De´glon et al., 2000; Hottinger et al., 2000; Kordower et al., 2000). GDNF is a survival factor for motoneurons against various insults. GDNF was reported to save 92% of facial motoneurons after neonatal axotomy of the facial nerve (Henderson et al., 1994; Yan et al., 1995), 50% of adult motoneurons lost after injuryinduced cell death (Li et al., 1995) and 50% of the facial motoneurons in mutant mice displaying progressive motor neuropathy (Sagot et al., 1996). Recently, we reported that the direct facial nucleus injection of lentivirus vector encoding the GDNF cDNA saved all the motoneurons that would normally die following a facial nerve axotomy in 4 months old Balb/C mice (Hottinger et al., 2000). Different studies have reported the administration of GDNF in the spinal cord of SOD1G93A transgenic mice using axonal retrograde transport though muscular injection of adenovirus or adeno-associated virus. Mohajeri et al. (1999) reported that intramuscular injection of myoblasts transduced by a retroviral vector encoding GDNF prevented the loss of large motoneurons and delayed the disease onset in SOD1G93A mutant mice but not their survival. Recently, Acsadi et al. (2002) also reported a significant delay in the onset of disease and a 2-week prolonged survival following the intramuscular injection of an adenovirus vector encoding GDNF. Similarly, Wang et al. (2002) showed a delay in disease onset and life span extension by an intramuscular administration of an adeno-associated virus vector (AAV) encoding GDNF in SOD1G93A mice. In the present report, we tested the protective effect of GDNF expressed specifically at the level of motoneuron cell bodies through the direct gene transfer in the spinal cord or facial nucleus of SOD1G93A transgenic mice using a lentiviral vector encoding GDNF to transduce neurons with con-

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sequent anterograde transport of the expressed protein throughout the cell bodies and axons. We report that the strong expression of GDNF at the lumbar level has no effect on diseased spinal motoneurons. In contrast, GDNF expression in the facial nucleus significantly rescued facial motoneurons from cell death. This difference may be explained by the fact that motoneuron loss is slower in facial nucleus compared to the spinal cord enhancing the ability of GDNF to act on motoneuron cell bodies. Our morphometric studies of motoneurons clearly underlined a cellular atrophy of remaining motoneurons in SOD1G93A transgenic mice that is more important in lumbar spinal cord compared to facial nucleus. This morphometric difference may to be explained by faster evolution and/or vulnerability in spinal motoneurons, explaining the rescue of facial motoneurons by lentivirus vector encoding GDNF. ALS is a multisystem degenerative disease, in which the earliest and most severe degenerative changes tend to affect lower and usually upper motoneurons. Even within motoneurons, there is selective vulnerability as subsets of motoneurons tend to be spared in ALS such as oculomotor nucleus in brainstem or the Onuf’s nucleus in sacral spinal cord. Several hypotheses ranging from different expression of Ca2+ binding proteins (calbindin and parvalbumin) in different types of motoneurons (Alexianu et al., 1994; Elliott and Snider, 1995; Morrison et al., 1998; Vanselow and Keller, 2000) or a selective resistance of motoneurons against an excitotoxic pathway (Reiner et al., 1995) may explain the differences of vulnerability. Another hypothesis relates to the existence of various molecular and neurochemical features explaining why facial motoneurons might be less vulnerable to insults (Shaw and Eggett, 2000). Spinal motoneurons are large cells with long axonal processes requiring higher mitochondrial activity compared to other motoneurons. The rationale behind this hypothesis is the observed vacuolar distortion of mitochondria occurring at an early stage in the cascade of motoneuron injury in SOD1 transgenic mice (Chiu et al., 1995; Kong and Xu, 1998; Wong et al., 1995). The long axons of spinal motoneurons necessitate an elaborate cytoskeleton explaining their very high neurofilament content compared to motoneurons with shorter axons such as the facial nucleus. Neurofilament subunits are assembled in the motoneuron cell body and are transported down the axon. The abnormal assembly and accumulation of neurofilaments in the cell body and proximal axons of motoneurons is a characteristic feature of ALS pathology and may lead to impairment of axonal transport. The axonal transport is composed by a fast anterograde transport used for transporting among others things vesicles of the constitutive secretory pathway, a slow anterograde transport responsible for the movement of cytoskeletal and soluble protein, and a retrograde axonal transport used to deliver neurotrophic signals and some viruses to neuronal cell bodies (Almenar-Queralt and Goldstein, 2001). Both a retrograde transport of GDNF from muscle consistent with its role in motoneuron survival and an anterograde transport of GDNF and its receptors have been reported in sensory and motoneurons, suggesting additional effects of GDNF in the peripheral region of motoneurons (Rind and Von Bartheld, 2002; Russell et al., 2000; Von Bartheld et al., 2001). Different studies have reported an impairment of both fast and slow anterograde axonal transport in SOD1G93A transgenic mice (Zhang et al., 1997; Warita et al., 1999; Williamson and Cleveland, 1999). The lack of behavioral effect observed in our study may be related to a poor GDNF transport to the gastrocnemius muscle, in spite of the strong

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expression of GDNF in the cell body. Moreover, Millecamps et al. (2001) demonstrated improved adenovirus gene transfer in the motoneurons of symptomatic ALS mice despite abnormalities in axonal transport reported in these mice and a plasticity of ALS motoneurons, which compensate for the loss of nerve fibers by acquiring new capacities for viral particle uptake compare to WT littermates motoneurons. All these different arguments could explain the different effect of GDNF in lumbar spinal cord between the intramuscular adenoviral injection and the soma lentivirus injection in the same animal model of ALS. From the present work and that related to intramuscular injections of either adenoviral or adeno-associated vectors encoding GDNF (Acsadi et al., 2002; Kaspar et al., 2003; Wang et al., 2002), the following conclusions can be made: GDNF exerts an obvious protective effect on both spinal and facial motoneurons in case of long-term intramuscular expression, whereas local GDNF delivery apparently leads to a protective effect only on facial motoneurons. GDNF therapeutic administration to motoneurons by adenoviral retrograde transport thus appears to be more efficient in SOD1G93A transgenic mice than lentiviral anterograde transport to protect cell body and axon of degenerescence. However, in a recent study using AAV muscle injection, Kaspar et al. (2003) have reported a superior effect of IGF-1 (insulin growth factor 1) versus GDNF in SOD1 transgenic mice suggesting that delivery to both the muscle and spinal cord is the most efficacious delivery method. The current understanding of the physiopathological mechanisms related to the disease may require the expression of intracellular acting proteins in motoneuron cell bodies (Lino et al., 2002). The present work demonstrates that the direct injection of lentiviral vectors allows long-term expression of intracellular proteins, a powerful property for screening potential therapeutics in mutated SOD1 transgenic mice.

Acknowledgments We thank Fabienne Pidoux and Maria Rey for lentiviral production and Dr. William Pralong for advice. This work was supported in part by the Mauro Baschirotto Rare Diseases Foundation and the Swiss National Science Foundation.

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