Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or β-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4–20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.
Parkinson's disease (PD) is a progressive neurodegenerative disorder that predominantly affects dopaminergic (DA) neurons in the substantia nigra (SN). Currently available therapies aim at replacing dopamine in the striatum to restore motor functions. However, it is essential to develop therapeutic interventions that block or slow down the ongoing degenerative process.1 Glial cell line-derived neurotrophic factor (GDNF) is the most potent neurotrophic factor discovered to date for DA neurons, and has been shown to enhance DA neuron survival both in vitro and in vivo, thus raising hopes that it may be valuable clinically.234 Administration of GDNF protein in the central nervous system is limited by its short activity and inability to cross the blood–brain barrier. Direct in vivo gene transfer therefore offers a more efficient method of GDNF delivery in a manner whereby the factor is continuously synthesised in the desired anatomical location.
Previous studies have demonstrated that GDNF gene delivered via adeno-associated viral (AAV), adenoviral (Ad) or lentiviral vectors protects nigral DA neurons while regenerating the nigrostriatal pathway in rodent and primate models of PD when administered before or shortly after an injection of neurotoxin.56789101112 However, the effectiveness of chronically delivered GDNF gene via AAV vector in a later phase of the degenerative process has not previously been documented. PD is a disorder characterized by progressive DA degeneration, and substantial numbers of DA neurons are depleted before the obvious appearance of symptoms. It is thus clinically more important to address whether GDNF gene delivered in a delayed manner is capable of rescuing the DA neurons and improving behavioral performance in animal models with extensive nigrostriatal DA denervation already present. In addition, it is not clear whether vector-derived GDNF could be retrogradely transported from axon terminals to cell bodies of DA neurons in the SN.
The present study examined the effect of AAV-mediated GDNF gene delivery into the striatum 4 weeks after creation of an intrastriatal lesion. Using AAV vector expressing FLAG peptide-tagged GDNF (AAV-GDNFflag), we found that transgene-derived GDNFflag was retrogradely transported to the SN and could halt the ongoing degeneration of nigrostriatal DA neurons, with functional recovery even after substantial numbers of DA cells had been lost.
AAV-GDNFflag expresses functional GDNFflag fusion protein in vitro
We constructed AAV vectors expressing FLAG-tagged GDNF (AAV-GDNFflag) or β-galactosidase (AAV-LacZ) (Figure 1). On Western blot analysis, GDNFflag fusion protein was detected in the lysate of AAV-GDNFflag-transduced 293 cells by both anti-GDNF and anti-FLAG antibodies. No signals were detected in the lysate of AAV-LacZ-transduced cells (Figure 2a).
The biological activity of the GDNFflag was examined in primary embryonic DA neuron cultures. Tyrosine hydroxylase (TH) immunostaining revealed that about twice as many TH-immunoreactive (TH-IR) cells survived in AAV-GDNFflag transduced cultures than in AAV-LacZ transduced cultures Figure 2b. These results confirmed that AAV-GDNFflag could drive production of the bioactive GDNFflag fusion protein in vitro, and that the addition of the FLAG peptide did not alter the neurotrophic effects of GDNF on DA neurons, allowing identification of exogenously expressed GDNFflag fusion protein by anti-FLAG antibody.
Expression of GDNFflag in the striatum and retrograde transport to SN
GDNFflag transgene expression was detected in the striatum with anti-FLAG antibody at 2, 4, 8 and 20 weeks after AAV-GDNFflag injection. FLAG immunoreactive (FLAG-IR) cells were detected around the injection sites throughout the striatum. These cells displayed the typical morphology of medium spiny neurons (Figure 3a, b). Furthermore, dual immunofluorescence staining with anti-TH and anti-FLAG antibodies detected double-positive cells in the pars compacta of ipsilateral SN at 4 and 8 weeks after intrastriatal AAV-GDNFflag injection Figure 3c–e. At 8 weeks after AAV vector injection, 13.1 ± 2.4% (n = 4) of TH-IR cells overlapped with FLAG-IR cells dispersed through the SN, indicating some transgenic GDNFflag obtained access to DA neurons. No FLAG-IR cells were observed in the striatum or SN on the contralateral side, nor in these regions in AAV-LacZ or vehicle-injected rats. In AAV-LacZ-injected rats, although X-gal-positive cells were detected in the striatum from 2–20 weeks after injection, no β-galactosidase signals could be detected in SN (data not shown). These results indicate that AAV-GDNFflag is capable of driving the expression of GDNFflag fusion protein in the striatum, and that GDNFflag fusion protein, not the AAV vector itself, could be retrogradely transported to the DA neuron bodies in SN from terminals in the striatum.
Rescue of DA innervation in the striatum
In order to determine whether AAV-GDNFflag would rescue DA neuron innervation in the lesioned striatum, the extent of TH-IR fiber terminals was examined on slices throughout the striatum at 300-μm intervals at 20 weeks after injection. In AAV-LacZ-injected rats, the density of TH-IR fibers in the lesioned striatum was remarkably reduced to only 13.5 ± 3.0% of the intact side (Figure 4a). In contrast, in rats injected with AAV-GDNFflag, the density of TH-IR fibers was significantly greater, reaching nearly 48.7 ± 7.3% of the intact side Figure 4b, d. Maintained TH-IR fibers were distributed around the injection sites, indicating that innervation of TH-IR fiber was rescued by the GDNFflag expressed in the striatum.
Maintenance of nigrostriatal projections
Cholera toxin subunit B (CTB, neuronal tracer)13 was injected bilaterally into the striatum to assess the extent of the remaining nigrostriatal pathway. The same coordinate of the 6-OHDA injection was utilized 3 days before death, to retrogradely label a subpopulation of DA neurons maintaining projections to the striatum. At the time point of AAV vector treatment, ie 4 weeks after 6-OHDA injection, the number of CTB-positive neurons in the lesioned SN was 28.9 ± 3.7% of the intact contralateral side (n = 5). Double immunofluorescence with anti-CTB and anti-TH antibodies revealed that most large CTB-positive neurons in SN were also TH-positive (Figure 5). The percentage of TH-IR neurons on the lesioned side was 34.9 ± 2.6% (n = 4) of the intact side. These results demonstrate that some portions of nigrostriatal connections remained at 4 weeks after lesion. At 20 weeks after the AAV vector injection, extensive loss of TH-IR cells (19.5 ± 2.0% of intact side) and CTB-positive cells (17.9 ± 1.6% of intact side) was observed in rats injected with AAV-LacZ. In contrast, injection of AAV-GDNFflag resulted in a significant increase in TH-IR neurons and CTB-positive cells, reaching 57.3 ± 6.2% and 50.8 ± 8.5%, respectively, of the contralateral side (Figure 6, P < 0.01). These results demonstrate that intrastriatal AAV-GDNFflag injection at 4-weeks post-lesion rescued the survival of DA neurons in the SN and promoted the maintenance of nigrostriatal projections.
Behavioural recovery after intrastriatal injection of AAV-GDNFflag
Four weeks after 6-OHDA administration, all rats in the three groups (AAV-GDNFflag, n = 20; AAV-LacZ, n = 16; and vehicle, n = 8) demonstrated a similar degree of impairment both in the apomorphine-induced rotation tests and in the cylinder tests, suggesting equivalent levels of DA depletion.
A decrease in apomorphine-induced rotations was observed in rats receiving AAV-GDNFflag, beginning at 4 weeks after the vector injection (Figure 7a). This decrease proceeded gradually and persisted throughout the experiment (20 weeks). In contrast, rats in the AAV-LacZ or vehicle injection group demonstrated a fairly stable rotation rate during the same period.
The cylinder test was used to measure spontaneous forelimb use in lesioned rats Figure 7b. Before AAV treatment, rats in each group showed a similar marked ipsilateral bias after a 6-OHDA lesion, as indicated by a reduced frequency of contralateral limb use (23–25% of total contacts) in this test. In rats receiving AAV-GDNFflag, significant improvement was observed from 4 weeks after injection, developing gradually and reaching near normal levels (47% of total contacts) at 20 weeks. In contrast, contralateral use persisted at the same level throughout the 20 weeks in rats receiving AAV-LacZ or vehicle. Statistical analysis of AAV-GDNFflag injected rats versus AAV-LacZ or vehicle-injected rats for both apomorphine-induced rotation and cylinder test revealed significant differences (P < 0.01).
Preservation of dopamine content in the lesioned striatum
To test whether the behavioral recovery following AAV-GDNFflag injection correlated with nigrostriatal dopamine production, dopamine levels and those of its metabolites in the striatum, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), were assayed at 20-weeks after injection. In AAV-GDNFflag-injected group (n = 8), the dopamine level on the lesioned side of the striatum was 50.9 ± 7.7% of the contralateral intact side, while in the AAV-LacZ injected group (n = 8) it remained at 11.5 ± 4.8% of the contralateral side (Figure 8, P < 0.01). In addition, levels of HVA and DOPAC were higher in the AAV-GDNFflag-injected group. No significant differences in levels of dopamine or its metabolites were observed on the intact side of striatum between the two groups.
The present results indicate that AAV vector-mediated delivery of GDNF gene into the striatum following a 6-OHDA lesion protects SN neurons from further late degeneration, while maintaining their connection to the striatum.
AAV vector is one of the most attractive gene delivery vehicles for direct introduction of therapeutic genes into the brain in the treatment of neurological diseases. Its unique characteristics include the lack of any disease associated with the wild-type virus, an ability to infect non-dividing cells, long-term transgene expression without a substantial immune response, and the physical stability of viral particles.14 Previous studies, including our own, have demonstrated that AAV vector-mediated delivery of dopamine-synthesizing enzymes results in efficient long-term expression of the transgene in the striatum.141516
The present study used the Sauer and Oertel partial PD model.17 In this model, intrastriatal injection of 6-OHDA induces progressive retrograde degeneration of DA neurons that starts between 1 to 2 weeks after lesioning and continues over 8 to 16 weeks. This ongoing depletion of DA neurons may be more similar to the disease process of PD and more appropriate as an animal model for therapeutic study than the complete model, which is constructed by destroying the medial forebrain bundle, thereby causing more rapid degeneration of DA neurons. In our experiment, rats had exhibited consistent behavioral deficits (>7 apomorphine-induced rotations/min) before vector injection. The appearance of apomorphine-induced rotations is generally assumed to represent ≥90% depletion of striatal dopamine content.18 However, studies on PD patients and animal models have indicated that there might be more surviving DA neurons than the levels of dopamine suggested.192021 In our model, the number of CTB-positive neurons on the lesioned side of SN was 28.9% of contralateral value at 4 weeks post-lesion. This is consistent with the previous studies using Fluorogold (FG)-retrograde labelling that demonstrated 28.8% (35 days post-lesion)bib: or 34% (4 weeks post-lesion)bib: of FG-positive cells in the lesioned SN. In addition, most CTB-labeled neurons were TH-positive, suggesting that part of the nigrostriatal projection remained intact at the time of AAV vector injection. These remaining portions of intact nigrostriatal projections and DA neurons may serve as substrate for regeneration and functional recovery after GDNF gene delivery.
CTB back-labelling is more accurate in the assessment of nigrostriatal function than anti-TH immunostaining alone, because CTB labels only the SN neurons that maintain functional nigrostriatal projections, while anti-TH immunostaining merely demonstrates the presence of TH enzyme in neuron bodies. In the AAV-GDNFflag group, more CTB-labeled DA neurons existed than in the SN, suggesting that GDNFflag expressed in the striatum could rescue DA neurons and promote functional projections to the striatum. In addition, successful rescue of the nigrostriatal system by intrastriatal AAV-GDNFflag injection was supported by high levels of dopamine and its metabolites in the striatum, indicating long-lasting activation of dopamine in the nigrostriatal system.
Intranigral and intrastriatal injections of GDNF protein or GDNF-expressing vectors have been reported to protect nigral cell bodies almost completely if the treatment is initiated before or shortly after insult.56789101112 However, when GDNF is administered with a delay after the insult, sparing of DA neurons is only marginal and the magnitude of functional recovery probably reflects the number of DA neurons still surviving and maintaining nigrostriatal connections.10232425 In contrast with previous studies1026 in which three or four deposits of 6-OHDA were injected to create more extensive striatal lesions, 6-OHDA was injected at only one site in our model and less damage might have been incurred to nigrostriatal connections. Using adenoviral vectors Kozlowski et al22 demonstrated that delivering GDNF gene to the SN 1 week after lesioning rescued DA neurons and increased the number of DA neurons maintaining a connection to the striatum. Conversely, expression of GDNF in the striatum did not exhibit significant ameliorative effects at 35 days post-lesion. The discrepancy between their result and ours with regards to behavioral recovery may reflect a longer survival time (20 weeks versus 35 days) of our rats. Striatal fibers may recover more slowly to provide delayed initiation and protracted time course of functional amelioration.10 Another possible explanation for the discrepancy is our use of multiple injection sites of AAV vectors. Given that limited diffusion of AAV vectors was demonstrated with transduced cells present no further than 1–2 mm from the injection site in the rodent central nervous system,272829 we injected AAV-GDNFflag at three different sites in the striatum to provide a broader transduction, particularly in the area distant from the site of 6-OHDA injection. Broad distribution of GDNFflag protein in the striatum may be essential for effective recovery of remaining neurons.
As 6-OHDA lesions or injections of apomorphine induce endogenous expression of GDNF,3031 it is necessary to distinguish transgene-derived GDNF from endogenous GDNF to evaluate the effect of gene delivery. Thus, we constructed an AAV vector that expresses FLAG-tagged GDNF. Although the protective effect on SN neurons after the intrastriatal delivery of GDNF could be explained by retrograde transport of GDNF from the striatum to the SN, no evidence has been demonstrated supporting this mechanism. In the present study, we detected the GDNFflag in SN cells ipsilateral to the AAV-GDNFflag injection side from 4–8 weeks after injection. These SN cells were confirmed to be DA cells using double immunofluorescent staining with anti-FLAG and anti-TH antibodies. FLAG-IR cells were observed in a broad area of the pars compacta at 8 weeks after AAV vector treatment, although the percentage of FLAG-IR cells seemed low (13.1% of TH-IR cells). As GDNF exhibits extremely potent effects in the picomolar range on DA neurons, small amounts of GDNFflag protein transported transiently and not detected by immunohistochemistry at the time of death might protect nigral DA neurons against toxin-induced cell death. In contrast, rats injected with AAV-LacZ in the striatum demonstrated no β-galactosidase signal in the SN. In agreement with the previous study,bib: AAV vector per se was not retrogradely transported in the central nervous system, but GDNFflag protein was retrogradely transported from the striatum to the DA neurons in the SN. We also detected substantial expression of GDNFflag protein in the striatum at 20 weeks after injection. This persistence of GDNFflag production provided long-term rescue of the nigrostriatal system after a single AAV vector injection. The density of TH-IR fibers in the lesioned striatum was preserved after the AAV-GDNFflag injection. This preservation of TH-immunoreactivity may be explained in part by the enhanced expression of TH in the remaining fibers. Increased levels of TH mRNA were demonstrated in SN neurons after the striatal delivery of GDNF gene.bib: Alternatively, stimulation of axonal sprouting in the GDNF-treated striatum may have occurred,1026 although the ability of GDNF to induce regeneration or sprouting of DA nerve terminals following a 6-OHDA lesion remains controversial.3334
Behavioral recovery persisted throughout the experiment without deterioration. However, motor asymmetry could not be completely abolished. Although transgene-derived GDNFflag rescued a substantial number of DA neurons from progressive degeneration, part of DA axon terminals may have already been lost at the time of GDNFflag expression, leading to irreversible degeneration of such severely damaged DA neurons. In addition, another possibility is to suggest that further functional recovery of lesioned nigrostriatal pathways is not obtained by GDNF alone, and that other neurotrophic factors such as neurturin or artemin, another member of GDNF family with strong homology to GDNF, may be necessary.353637
In conclusion, we have shown that (1) AAV vector-mediated GDNF gene delivery in the striatum promoted the survival of DA neurons in the SN, increased the density of TH-IR fibers, and ameliorated behavioral and biochemical deficits even after the degenerative process had begun; (2) GDNFflag fusion protein was detected in the SN, suggesting the transgene-derived GDNF was retrogradely transported from the striatum to DA cell bodies in the SN, while maintaining functional connections within the nigrostriatal pathway. These data indicate that AAV-mediated GDNF gene delivery in the striatum is feasible as a neuroprotective gene therapy of PD.
Materials and methods
Recombinant AAV vector production
AAV vector plasmid pAAV-GDNFflag was derived from previously described pAAV-GDNF plasmid.bib: This plasmid contains the mouse GDNF cDNAbib: tagged by FLAG sequence (DYKDDDDK) at the carboxyl terminus under the human cytomegalovirus (CMV) immediate–early promoter, with human growth hormone first intron and simian virus 40 (SV40) polyadenylation signal sequence between the inverted terminal repeats (ITR) of the AAV-2 genome. AAV vector plasmid pAAV-LacZ, auxiliary plasmid pHLP19 and pladenol were described previously.bib: Subconfluent human 293 cells were transiently transfected with vector plasmid and helper plasmid using the calcium phosphate co-precipitation method. Seventy-two hours after transfection, cells were harvested and lysed by three freeze and thaw cycles. AAV vectors (AAV-GDNFflag and AAV-LacZ) were purified using two sequential continuous CsCl gradients, as described previously.bib: The final particle titer of the AAV-GDNFflag was 1.6 × 1013 vector genome copies/ml and AAV-LacZ was 2.1 × 1013 vector genome copies/ml, as estimated by quantitative DNA dot-blot hybridization analysis.
In vitro expression of AAV-GDNFflag
To detect the in vitro expression of GDNFflag fusion protein, 293 cells were transduced with AAV-GDNFflag or AAV-LacZ (5000 vector genome copies/cell). Thirty-six hours after transduction, cell lysates were separated by 15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Residual protein-binding sites were blocked with 5% non-fat dried milk in TBS for 1 h at room temperature. Membranes were incubated overnight at 4°C using mouse anti-FLAG M2 antibody (1:1000, Sigma, St Louis, MO, USA) or rabbit anti-GDNF antibody (1:2000, Santa Cruz, Santa Cruz, CA, USA), followed by incubation with a secondary anti-mouse or anti-rabbit conjugated to horseradish peroxidase (1:2500, Amersham, Arlington Heights, IL, USA) for 2 h at room temperature. Chemiluminescent signals were detected by the ECL systems (Amersham).
To assay the biological activity of AAV vector-derived GDNFflag fusion protein, primary cultures of rat fetal DA neurons were prepared. Briefly, the ventral mesencephalon was dissected out from day 14 Wistar rat embryos, minced and incubated with 0.25% trypsin in PBS for 15 min. Isolated cells were plated in 35-mm dishes pre-coated with poly-L-lysine, at an initial cell density of 105 cells/dish, and incubated at 37°C with Neurobasal-SFM Media (Gibco BRL) supplemented with B27 (Gibco BRL), 100 units/ml penicillin, 100 μg/ml streptomycin, 25 mM KCl, and 2 mM glutamine. In AAV vector-treated dishes, cultures were infected with AAV-GDNFflag or AAV-LacZ (5000 vector genome copies/cell). In the positive control dish or negative control dish, recombinant human GDNF (rhGDNF, Santa Cruz) or BSA was added to cultures at a final concentration of 10 ng/ml. Culture medium was refreshed by half fresh medium at 3-day intervals, with rhGDNF or BSA kept at the same concentration. After 9 days, cells were fixed with 4% paraformaldehyde (PFA) for 30 min and processed for immunocytochemical staining to examine the expression of GDNFflag and TH. The number of TH-IR cells present was counted as described.bib:
Animals and surgical procedures
Adult male Wistar rats (weight 200 to 250 g) were maintained in a 12-h light/dark cycle in cages with ad libitum access to food and water. Experiments were conducted in accordance with the Jichi Medical School guidelines for animal care. The partial rat model of PD was prepared as described by Sauer and Oertel.bib: Briefly, rats received stereotactic injections of 20 μg 6-hydroxydopamine (6-OHDA; calculated as free base; Sigma) dissolved in 4 μl of ascorbate-saline (0.05%) in the left striatum at the following coordinates: anterior-posterior (AP) +1.0 mm, medial-lateral (ML) 3.0 mm, dorso-ventral (DV) −4.5 mm. The stereotactic coordinates were calculated relative to the bregma and dural surface. After 4 weeks, animals were tested for apomorphine-induced rotation (0.1 mg/kg administered intraperitoneally) and only those animals exhibiting seven or more contralateral rotations/min in a 60-min period were included in further study. To assess the extent of nigrostriatal degeneration, neural tracer CTB (1% in distilled H2O, 1 μl; List Biological Laboratories, Campbell, CA, USA) was injected bilaterally into the striatum (n = 5, AP = + 1 mm, ML = 3.0 mm, DV = −4.5 mm) 3 days before death. This was performed to retrogradely label the subpopulation of DA neurons in the SN maintaining functional projections to the striatum at 4 weeks after 6-OHDA lesion. PD animal models were randomly assigned to receive AAV-GDNFflag (n = 32), AAV–LacZ (n = 22), or vehicle (0.1 M PBS; n = 8) injection in the left striatum at three different sites (2 μl per site, 1013vector genome copies/ml) using the following coordinates: (AP = + 1.5 mm, + 1.0 mm and + 0.5 mm; ML = 2.6 mm, 3.0 mm and 3.2 mm; DV = −4.5 mm). The injection rate was set at 1 μl/min and the needle was left in place for an additional 7 min before being slowly retracted. To evaluate retrograde transport of GDNFflag protein, some rats (at each time point: AAV-GDNFflag-injected group, n = 4; AAV-LacZ-injected group, n = 2) were killed for histology at 2, 4 and 8 weeks after AAV injection, respectively. Twenty weeks after vector injection, rats were randomly grouped and killed for biochemical or immunohistochemical analysis. In eight rats, CTB was injected bilaterally into the striatum 3 days before death.
After AAV vector injection, rats were tested for rotational behavior every 2 weeks by intraperitoneal injection of apomorphine-HCl (0.1 mg/kg). The total number of complete body turns was counted during an observation period of ≥60 min, as described previously.16 Spontaneous limb use was scored every 4 weeks using the cylinder test method.10 Rats were placed in a clear glass cylinder large enough for free movement. After they had performed 10 rears during which they placed at least one paw on the cylinder wall, the number of both forepaw contacts to the wall of the cylinder was counted at least 20 times. The data for contralateral forepaw contacts are given as a percentage of total.
The rats were killed by decapitation under sodium pentobarbital anesthesia and brains were immediately dissected and placed on dry ice. The striatum was punched out bilaterally using a sharp-edged, stainless steel tube. Wet tissue samples were weighed and stored at −80°C until subsequent analysis. Levels of dopamine and its metabolites, HVA and DOPAC, were estimated using high-performance liquid chromatography (HPLC) as described previously.bib:
Rats were perfused with PBS under deep anesthesia followed by ice-cold 4% PFA. Brains were dissected, and then post-fixed for 4 h in 4% PFA. Coronal sections (30 μm) from the striatum and SN were treated for 30 min in 0.3% H2O2 in PBS and blocked with 3% fetal bovine serum (FBS) in PBS/0.1% Triton X-100 for 1 h. Sections were then incubated with primary antibodies for TH (1:1000, mouse anti-TH monoclonal antibody; Chemicon, Temecula, CA, USA), CTB (1:10 000, goat antiserum to CTB; List Biologic, Campbell, CA, USA) or FLAG (1:1000, anti-FLAG M2 antibody, Sigma) at 4°C overnight. This was followed by incubation with biotinylated secondary antibodies (to species of primary antibodies) for 1 h at room temperature (1:400, Santa Cruz). Sections were visualized with avidin-biotinylated peroxidase complex procedure (Vectastain ABC kits; Vector Laboratories, Burlingame, CA, USA) using 3,3-diaminobenzidine (DAB) as a chromogen.
For FLAG/TH double immunofluorescence staining, sections were sequentially incubated with blocking solution containing 5% normal goat serum in PBS for 1 h at room temperature, monoclonal mouse anti-FLAG antibody (1:250, Sigma) and polyclonal rabbit anti-TH antibody (1:500) overnight at 4°C, and rhodamine-conjugated goat anti-mouse IgG (1:200; Santa Cruz) and FITC-conjugated goat anti-rabbit IgG (1:200; Santa Cruz) for 2 h at room temperature. Sections were examined and viewed under confocal laser scanning microscopy (TCS NT; Leica, Heidelberg, Germany). For CTB/TH double-immunofluorescence staining, anti-CTB (1:5000, goat antiserum to CTB) and mouse anti-TH (1:500) antibodies were used.
For quantitative analyses of TH-positive neurons and CTB-labeled neurons, sections at 300-μm intervals throughout the SN bilaterally were counted. Optical density of TH-IR fibers was quantified on sections every 300 μm rostro-caudally throughout the striatum using NIH Image 1.59 software.bib:
Results are presented as mean ± s.e.m. Data were statistically analyzed using repeated-measures analysis of variance (ANOVA) followed by Tukey honestly significance difference (HSD) test (StatView 5.0 software).
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We thank Masashi Urabe and Dongsheng Fan for helpful advice. We also thank Avigen for providing the AAV vector production system. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government; by Health Sciences Research Grants from the Ministry of Health Labour and Welfare of Japan; and by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST).
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Wang, L., Muramatsu, S., Lu, Y. et al. Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease. Gene Ther 9, 381–389 (2002) doi:10.1038/sj.gt.3301682
- adeno-associated virus
- glial cell line-derived neurotrophic factor
- Parkinson's disease
- gene therapy
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