Research Article

Neurodegeneration Prevented by Lentiviral Vector Delivery of GDNF in Primate Models of Parkinson's Disease

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Science  27 Oct 2000:
Vol. 290, Issue 5492, pp. 767-773
DOI: 10.1126/science.290.5492.767

Abstract

Lentiviral delivery of glial cell line–derived neurotrophic factor (lenti-GDNF) was tested for its trophic effects upon degenerating nigrostriatal neurons in nonhuman primate models of Parkinson's disease (PD). We injected lenti-GDNF into the striatum and substantia nigra of nonlesioned aged rhesus monkeys or young adult rhesus monkeys treated 1 week prior with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lenti-GDNF augmented dopaminergic function. In MPTP-treated monkeys, lenti-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lenti-GDNF injections to intact rhesus monkeys revealed long-term gene expression (8 months). In MPTP-treated monkeys, lenti-GDNF treatment reversed motor deficits in a hand-reach task. These data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in primate models of PD and might be a viable therapeutic strategy for PD patients.

Parkinson's disease is a progressive disorder resulting from degeneration of dopaminergic neurons within the substantia nigra. Surgical therapies aimed at replacing lost dopaminergic neurons or disrupting aberrant basal ganglia circuitry have recently been tested (1). However, these clinical trials have focused on patients with advanced disease, and the primary goal of forestalling disease progression in newly diagnosed patients has yet to be realized.

Glial cell line–derived neurotrophic factor (GDNF) has potent trophic effects on dopaminergic nigral neurons (2–8), suggesting that this factor could provide neuroprotection in patients with early PD. We have shown that intraventricular administration of GDNF failed to improve clinical function or prevent nigrostriatal degeneration in a patient with PD, and this failure resulted from an ineffective delivery method (9). Gene therapy is a powerful means to deliver trophic molecules to the central nervous system in a site-specific manner. Robust transfer of marker and therapeutic genes has recently been demonstrated in the rodent and nonhuman primate brain with the use of a lentiviral vector (10–15). The transgene expression is long-term and nontoxic. Using two different nonhuman primate models of PD, we examined whether lentiviral-mediated delivery of GDNF could reverse the cellular and behavioral changes associated with nigrostriatal degeneration in primates. For the first model, we chose nonlesioned aged monkeys that displayed a slow progressive loss of dopamine within the striatum and tyrosine hydroxylase (TH) within the substantia nigra without frank cellular degeneration (16). These aged monkeys demonstrate changes within the nigrostriatal system that model some of the incipient cellular changes seen in early PD (17). In the second model, young adult monkeys received unilateral intracarotid injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce extensive nigrostriatal degeneration, resulting in a behavioral syndrome characterized by robust motor deficits.

In the first experiment, eight aged (approximately 25 years old) female rhesus monkeys received injections of lentiviral vectors encoding β-galactosidase (lenti-βGal; n = 4) or GDNF (lenti-GDNF; n = 4) targeted for the striatum and substantia nigra (18) and were killed 3 months later. Postmortem, all GDNF injections were localized to the caudate nucleus, putamen, and supranigral regions (19), as revealed by standard staining procedures (20). All aged monkeys receiving lenti-GDNF displayed robust GDNF immunoreactivity within the right striatum (Fig. 1A) and substantia nigra (Fig. 1C). In contrast, no monkeys receiving lenti-βGal displayed specific GDNF immunoreactivity in the right striatum (Fig. 1B). Rather, these monkeys displayed robust expression of βGal similar to that reported previously (15). In lenti-GDNF–treated animals, GDNF immunoreactivity within the striatum was extremely dense and distributed throughout the neuropil (Fig. 1). When the primary antibody concentration was decreased to one-tenth of the standard, the intense striatal neuropil staining was diminished, and GDNF-immunoreactive perikarya were easily seen. Numerous GDNF-immunoreactive perikarya were also seen within the substantia nigra of lenti-GDNF–injected monkeys. Within the striatum and substantia nigra, Nissl-stained sections revealed normal striatal cytoarchitecture without significant cytotoxicity. Macrophages were occasionally observed within the needle tracts. Gliosis was similar across treatment groups and was principally confined to the regions immediately surrounding the needle tracts.

Figure 1

(A) Dense GDNF immunoreactivity within the head of the caudate nucleus and putamen in a lenti-GDNF–treated aged monkey. (B) In contrast, no GDNF immunoreactivity was observed in these regions in a lenti-βGal–treated animal. IC, internal capsule. (C) Dense GDNF immunoreactivity was observed within the midbrain of a lenti-GDNF–treated animal. (D) GDNF immunoreactivity within the forebrain of a lenti-GDNF–treated monkey. The staining within the putamen (Pt) is from an injection site. The staining within both segments of the globus pallidus (GPe and GPi) is the result of anterograde transport. (E) Anterogradely transported GDNF was also seen in the substantia nigra pars reticulata. Note that the holes in the tissue sections were made post mortem for HPLC analysis. Asterisk in (E) represents a lenti-GDNF injection site (CP, cerebral peduncle). Scale bar in (D) represents 1600 μm for panels A, B, and D; 1150 μm for panel C, and 800 μm for panel E.

Lenti-GDNF injections resulted in marked anterograde transport of the trophic factor. Intense GDNF immunoreactivity was observed within fibers of the globus pallidus (Fig. 1D) and substantia nigra pars reticulata (Fig. 1E) after striatal injections. GDNF-containing fibers emanating from putaminal injection sites were seen coursing medially toward and into the globus pallidus (Fig. 1D). These staining patterns were clearly distinct from the injection site and respected the boundaries of the striatal target structures. In contrast, anterograde transport of βGal was not observed in lenti-βGal monkeys. This suggests that secreted GDNF, and not the virus per se, was anterogradely transported.

Aged monkeys underwent fluorodopa (FD) positron emission tomography (PET) before surgery and again just before being killed (21). Before treatment, all monkeys displayed symmetrical FD uptake in the caudate and putamen bilaterally (ratio: 1.02 ± 0.02) (Figs. 2A and 2B, left). Similarly, there was symmetrical (4% difference) FD uptake in all lenti-βGal–treated monkeys after lentivirus injections (Fig. 2A, right). In contrast, FD uptake was significantly asymmetrical (27%) in lenti-GDNF–treated monkeys with greater uptake on the side of the GDNF expression (P < 0.007; Fig. 2B, right). With respect to absolute values, lenti-βGal animals displayed a trend toward reduced FD uptake after treatment relative to baseline levels (P= 0.06). Qualitatively, three of four lenti-GDNF–treated monkeys displayed clear increases in FD uptake on the treated side. This increase in uptake (K i value) between the groups just failed to reach statistical significance (P = 0.06).

Figure 2

PET scan data evaluating the influence of lenti-GDNF on FD uptake in (A and B) intact aged monkeys and (C and D) young adult MPTP-treated monkeys. (A) FD uptake did not change from baseline to 3 months after lentivirus injection in lenti-βGal–treated aged monkeys. (B) In contrast, lenti-GDNF injections manifested increased FD uptake on the side of GDNF expression relative to preoperative levels in aged monkeys. K i values (per minute) for the striatum are as follows: (left side) lenti-βGal preoperative 0.0068 ± 0.0001, lenti-βGal postoperative 0.0062 ± 0.0002; (right side) lenti-βGal preoperative 0.0068 ± 0.0002, lenti-βGal postoperative 0.0065 ± 0.0001; (left side) lenti-GDNF preoperative 0.0072 ± 0.0005, lenti-GDNF postoperative 0.0068 ± 0.0003; (right side) lenti-GDNF preoperative 0.0076 ± 0.0004, lenti-GDNF postoperative 0.0081 ± 0.0003. (C) After MPTP lesions, a comprehensive loss of FD uptake was seen within the right striatum of lenti-βGal–treated young adult monkeys. (D) In contrast, FD uptake was enhanced in lenti-GDNF–treated monkeys. K i values (per minute) for the striatum are as follows: lenti-βGal left, 0.0091 ± 0.0004; lenti-βGal right, 0.0017 ± 0.0005; lenti-GDNF left, 0.0084 ± 0.0004; lenti-GDNF right, 0.0056 ± 0.0018.

Within the striatum, lentiviral delivery of GDNF increased a number of markers of dopaminergic function (22). Optical density measurements were performed to assess the relative intensity of TH staining within the caudate nucleus and putamen (Fig. 3, A and B). On the left side where there was no lenti-GDNF expression, the intensity of TH immunoreactivity within the caudate nucleus and putamen was similar between groups (Fig. 3, A and B). In contrast, significant increases in optical density measures of TH immunoreactivity were seen in the right striatum of lenti-GDNF–infused monkeys (Fig. 3A) relative to lenti-βGal–treated animals (Fig. 3B) or the contralateral side (Fig. 3A). In this regard, there was a 44.1% and a 38.9% increase in optical density measures of TH immunoreactivity within the caudate nucleus and putamen, respectively (Fig. 4D). At the time of death, tissue punches were taken throughout the caudate nucleus and putamen of all monkeys. Relative to lenti-βGal–treated animals, measurement of dopamine (DA) and homovanillic acid (HVA) revealed significant increases in the right caudate nucleus (140% DA,P < 0.001; 207% HVA, P < 0.001) and putamen (47.2% DA, P < 0.05; 128% HVA,P < 0.01) in lenti-GDNF–treated aged monkeys (Fig. 4, E and F).

Figure 3

(A) Section stained for TH immunoreactivity through the anterior commissure illustrating the increase in TH immunoreactivity within the right caudate nucleus and putamen after lenti-GDNF delivery to aged monkeys. (B) Symmetrical and less intense staining for TH immunoreactivity in a monkey injected with lenti-βGal. (C) There were greater numbers and larger TH-immunoreactive neurons within the substantia nigra of a lenti-GDNF–treated animal relative to (D) a lenti-βGal–treated monkey. (E) Lenti-GDNF–treated aged monkeys displayed increased TH mRNA relative to (F) lenti-βGal–treated monkeys in the SN. Scale bar in (F) represents 4500 μm for panels 250 μm for panels (C) and (D) and 100 μm for panels (E) and (F).

Figure 4

(A through F) Plots of quantitative data illustrating enhanced nigrostriatal function in lenti-GDNF–treated aged monkeys. Solid bars denote lenti-βGal–treated monkeys; hatched bars indicate lenti-GDNF–treated monkeys. GDNF expression was limited to the right striatum and nigra. **P < 0.01; ***P< 0.001.

Lentiviral delivery of GDNF to aged monkeys resulted in an increase in the number of TH-immunoreactive neurons within the substantia nigra (Fig. 3, C and D). Regardless of the extent of GDNF immunoreactivity within the midbrain, the organization of TH-immunoreactive neurons was similar in all animals, and these neurons were not observed in ectopic locations within this locus. Stereological counts revealed an 85% increase in the number of TH-immunoreactive nigral neurons on the side receiving lentivirally delivered GDNF (Fig. 4A) relative to lenti-βGal–treated animals. On the side (left) that did not display GDNF immunoreactivity, lenti-GDNF–treated animals contained 76,929 ± 4918 TH-immunoreactive neurons. This is similar to what was seen in lenti-βGal–infused animals (68,543 ± 5519). Whereas lenti-βGal–infused monkeys contained 63,738 ± 6094 TH-immunoreactive nigral neurons in the right side, lenti-GDNF–treated monkeys contained 118,170 ± 8631 TH-immunoreactive nigral neurons in this hemisphere (P< 0.001).

A similar pattern was seen when the volume of TH-immunoreactive substantia nigra neurons was quantified (Fig. 4B). TH-immunoreactive neurons from lenti-βGal– and lenti-GDNF–treated monkeys were similar in size in the left nigra where there was no GDNF expression (11,147.5 ± 351 μm3 and 11,458.7 ± 379 μm3, respectively). In contrast, a 35% increase in neuronal volume was seen on the GDNF-rich right side in lenti-GDNF–injected aged monkeys (lenti-βGal 10,707.5 ± 333 μm3; lenti-GDNF 16,653.7 ± 1240 μm3; P < 0.001).

Although stereological counts of TH mRNA–containing neurons were not performed, there was an obvious increase in the number of TH mRNA–containing neurons within the right substantia nigra in lenti-GDNF–treated monkeys (Fig. 3E) compared with lenti-βGal–containing animals (Fig. 3F). With regard to the relative levels of TH mRNA expression within individual nigral neurons (23), the pattern of results was similar to that observed with TH-immunoreactive neuronal number and volume (Fig. 4C). On the left side, the optical density of TH mRNA within nigral neurons was similar between lenti-βGal– and lenti-GDNF–treated monkeys (78.28 ± 2.78 and 80.58 ± 2.5, respectively). In contrast, there was a significant (21.5%) increase in the optical density for TH mRNA in lenti-GDNF–treated monkeys (98.3 ± 1.5) relative to lenti-βGal–treated monkeys (77.2 ± 2.3) on the right side (P < 0.01).

In the second experiment, 20 young adult rhesus were initially trained 3 days per week until asymptotic performance was achieved on a hand-reach task in which the time to pick up food treats out of recessed wells was measured (16, 24). Each experimental day, monkeys received 10 trials per hand. Once per week, monkeys were also evaluated on a modified parkinsonian clinical rating scale (CRS). All monkeys then received an injection of 3 mg MPTP-HCl into the right carotid artery, initiating a parkinsonian state. One week later, monkeys were evaluated on the CRS. Only monkeys displaying severe hemiparkinsonism with the classic crooked arm posture and dragging leg on the left side continued in the study (n= 10). It is our experience that monkeys with this behavioral phenotype display the most severe lesions neuroanatomically and do not display spontaneous recovery behaviorally (24). On the basis of CRS scores, monkeys were matched into two groups of five monkeys, which received on that day lenti-βGal or lenti-GDNF treatment. Using magnetic resonance imaging (MRI) guidance, we gave all monkeys lentivirus injections into the caudate nucleus (n = 2), putamen (n = 3), and substantia nigra (n = 1) on the right side using the same injection parameters as in experiment 1. One week later, monkeys began retesting on the hand-reach task three times per week for 3 weeks per month (25). For statistical analyses, the times for an individual week were combined into a single score. During the weeks of hand-reach testing, monkeys were also scored once per week on the CRS. Individuals blinded to the experimental treatment performed all behavioral assessments. Three months after lentivirus treatment, monkeys received a FD PET scan and were killed 24 to 48 hours later, and tissues were histologically processed as before.

Within 1 week after the lentivirus injections, one monkey from each group died. Necropsies from these animals revealed only the presence of mild necrosis from multifocal random hepatocellular coagulation. On account of these deaths, all remaining monkeys underwent detailed necropsies after death, and no significant abnormalities in any organs were seen.

Before MPTP treatment, all young adult monkeys scored 0 on the CRS. After MPTP, but before lentivirus injection, monkeys in the lenti-GDNF and lenti-βGal groups averaged 10.4 ± 0.07 and 10.6 ± 0.6, respectively, on the CRS (P > 0.05). After lentivirus treatment, significant differences in CRS scores were seen between the two groups (Kolmogorov-Smirnov test, P < 0.0001; Fig. 5A). CRS scores of monkeys receiving lenti-βGal did not change over the 3-month period after treatment. In contrast, CRS scores of monkeys receiving lenti-GDNF significantly diminished during the 3-month period after treatment. Scores began to decrease in the first month after lenti-GDNF treatment. However, statistically significant differences between lenti-GDNF and lenti-βGal were only discerned at posttreatment observations 6, 7, 8, and 9 (Kolmogorov-Smirnov test, P < 0.04 for each comparison).

Figure 5

After MPTP-treatment, lenti-GDNF–injected monkeys displayed functional improvement on (A) the clinical rating scale and (B) the hand-reach task. All tests were performed 3 weeks per month [see (15)]. On the clinical rating scale, monkeys were matched into groups based upon the post-MPTP score. For the hand-reach task, each symbol represents the mean of three sessions per week for the left hand. Monkeys were not tested on this task during the week between MPTP and lentivirus injection. *P < 0.05 relative to lenti-βGal.

Lenti-GDNF–treated animals also improved performance on the operant hand-reach task. Under the conditions before MPTP administration, animals in both groups performed this task with similar speed (Fig. 5B). For the “unaffected” right hand, no differences in motor function were discerned for either group relative to performance before MPTP administration or to each other (P > 0.05). In contrast, performance with the left hand was significantly improved in lenti-GDNF–treated animals relative to controls (P < 0.05). After MPTP, all lenti-βGal–treated animals were severely impaired, with monkeys often not performing at all, or requiring more than the maximally allowed 30 s. In contrast, three of the four lenti-GDNF monkeys performed the task with the left hand at near-normal levels, whereas one lenti-GDNF–treated monkey was impaired and performed this task in a manner similar to the lenti-βGal–treated animals. Between groups, significant differences in performance were discerned on posttreatment tests 4, 6, 7, 8, and 9 (P < 0.05 for each comparison).

Just before being killed, all monkeys underwent FD PET scans. Qualitatively, all lenti-βGal–treated monkeys displayed pronounced FD uptake in the left striatum and a comprehensive loss of FD uptake on the right side (Fig. 2C). In contrast, two of four lenti-GDNF–treated animals displayed robust and symmetrical FD uptake on both sides (Fig. 2D). The remaining two lenti-GDNF monkeys displayed reduced FD uptake on the right side, but with K i values 50 to 100% greater than lenti-βGal controls (Fig. 2). Quantitatively, no differences in FD uptake were observed between groups within the left striatum (P > 0.05). In contrast, there was a significant (>300%) increase in FD uptake in lenti-GDNF–treated animals in the right striatum relative to lenti-βGal–treated animals (P < 0.05). When the right striatum was subdivided, significant increases in FD uptake were only seen within the putamen of lenti-GDNF–treated animals (P < 0.05).

After death, a strong GDNF-immunoreactive signal was seen in the caudate nucleus, putamen, and substantia nigra of all lenti-GDNF–treated, but none of the lenti-βGal–treated animals. The intensity and distribution of GDNF immunoreactivity was indistinguishable from what we observed in aged monkeys (see Fig. 1).

All lenti-βGal–treated monkeys displayed a comprehensive loss of TH immunoreactivity within the striatum on the side ipsilateral to the MPTP injection (Fig. 6A). In contrast, all lenti-GDNF–treated monkeys displayed enhanced striatal TH immunoreactivity relative to βGal controls (Fig. 6B). However, there was variability in the degree of striatal TH immunoreactivity in lenti-GDNF–treated animals and that variability was associated with the degree of functional recovery seen on the hand-reach task. Two lenti-GDNF–treated monkeys displayed dense TH immunoreactivity throughout the rostrocaudal extent of the striatum (Fig. 6B). In these monkeys, the intensity of the TH immunoreactivity was greater than that observed on the intact side. These two animals displayed the best functional recovery. A third lenti-GDNF–treated monkey also displayed robust functional recovery on the hand-reach task. However, the enhanced striatal TH immunoreactivity in this animal was limited to the post-commissural putamen. The fourth lenti-GDNF–treated monkey did not recover on the hand-reach task. Although putaminal TH immunoreactivity in this animal was still greater than controls, the degree of innervation was sparse and restricted to the medial post-commissural putamen.

Figure 6

(A and B) Low-power dark-field photomicrographs through the right striatum of TH-immunostained sections of MPTP-treated monkeys treated with (A) lenti-βGal or (B) lenti-GDNF. (A) There was a comprehensive loss of TH immunoreactivity in the caudate and putamen of lenti-βGal–treated animal. In contrast, near normal level of TH immunoreactivity is seen in lenti-GDNF–treated animals. Low-power (C and D) and medium-power (E and F) photomicrographs of TH-immunostained section through the substantia nigra of animals treated with lenti-βGal (C and E) and lenti-GDNF (D and F). Note the loss of TH-immunoreactive neurons in the lenti-βGal–treated animals on the side of the MPTP-injection. TH-immunoreactive sprouting fibers, as well as a supranormal number of TH-immunoreactive nigral perikarya are seen in lenti-GDNF–treated animals on the side of the MPTP injection. (G and H) Bright-field low-power photomicrographs of a TH-immunostained section from a lenti-GDNF–treated monkey. (G) Note the normal TH-immunoreactive fiber density through the globus pallidus on the intact side that was not treated with lenti-GDNF. (H) In contrast, an enhanced network of TH-immunoreactive fibers is seen on the side treated with both MPTP and lenti-GDNF. Scale bar in (G) represents the following magnifications: (A), (B), (C), and (D) at 3500 μm; (E), (F), (G), and (H) at 1150 μm.

Lenti-GDNF treatment enhanced the expression of TH-immunoreactive fibers throughout the nigrostriatal pathway. Unlike what was observed in aged monkeys, however, some TH-immunoreactive fibers in the striatum displayed a morphology characteristic of both degenerating and regenerating fibers. Large, thickened fibers could be seen coursing in an irregular fashion in these animals. Rostrally, these fibers appeared disorganized at times, with a more normal organization seen more caudally. TH-immunoreactive sprouting was also seen in the globus pallidus (Fig. 6, G and H), substantia innominata (Fig. 6, A and B), and lateral septum. These novel staining patterns were not immunoreactive for dopamine β-hydroxylase confirming the dopaminergic phenotype of this response.

Quantitatively, lenti-βGal–treated monkeys displayed significant decreases in the optical density of TH-immunoreactive fibers within the right caudate nucleus (71.5%; P < 0.006; Fig. 7D) and putamen (74.3% P< 0.0007; Fig. 7D) relative to the intact side. When analyzed as a group, TH optical density in the right caudate nucleus and putamen of lenti-GDNF–treated monkeys was significantly greater than that seen in lenti-βGal–treated monkeys (P < 0.001 for both) and was similar to that seen on the intact side of these animals (P > 0.05 for both).

Figure 7

(A through D) Quantification of lenti-GDNF's trophic effects on nigral neuronal number, volume, TH mRNA and striatal TH immunoreactivity in MPTP-treated monkeys. ***P < 0.001 significant decreases relative to intact side; ttt denotes significant increases relative to the intact side.

All lenti-βGal–treated monkeys displayed a dramatic loss of TH-immunoreactive neurons within the substantia nigra on the side ipsilateral to the MPTP injection (Fig. 7A). In contrast, the nigra from all four of the lenti-GDNF–treated displayed complete neuroprotection (Fig. 7A), regardless of the degree of functional recovery. In lenti-βGal–treated monkeys, intracarotid injections of MPTP resulted in an 89% decrease in the number (Fig. 7A), and an 81.6% decrease in the density, of TH-immunoreactive nigral neurons on the side ipsilateral to the toxin injection (P < 0.001). In contrast, lenti-GDNF–treated monkeys displayed 32% more TH-immunoreactive nigral neurons (P < 0.001) and an 11% increase in TH-immunoreactive neuronal density (P< 0.05) relative to the intact side. In lenti-βGal–treated animals, MPTP significantly reduced (32%) the volume of residual TH-immunoreactive nigral neurons on the lesion side relative to the intact side (P < 0.001; Fig. 7B). In contrast, the volume of TH-immunoreactive neurons in lenti-GDNF–treated animals was significantly larger (44.3%) on the lesioned side relative to the intact side (P < 0.001). Finally, the optical density of TH mRNA was quantified bilaterally in all animals (Fig. 7C). In lenti-βGal–treated animals, there was a significant decrease (24.0%) in the relative optical density of TH mRNA within residual neurons on the MPTP-lesioned side relative to the intact side (P < 0.03). In contrast, lenti-GDNF–treated animals displayed a significant increase (41.7%) in relative optical density of TH mRNA relative to the intact side or lenti-βGal–treated animals (P < 0.001).

Sections from all monkeys were stained for CD45, CD3, and CD8 markers to assess the immune response after lentiviral vector injection (26). These antibodies are markers for activated microglia, T cells, and leukocytes including lymphocytes, monocytes, granulocytes, eosinophils, and thymocytes. Staining for these immune markers was weak, and often absent, in these animals. Mild staining for CD45 and CD8 was seen in two animals. Some CD45-immunoreactive cells displayed a microglial morphology. Other monkeys displayed virtually no immunoreactivity even in sections containing needle tracts.

Two additional intact young adult rhesus monkeys received lenti-GDNF injections into the right caudate and putamen and the left substantia nigra using the same injection protocol (26). These animals were killed 8 months later and were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) (27) for long-term gene expression. Robust GDNF immunoreactivity was seen in the right caudate, right putamen, and left ventral midbrain in both animals. In the right substantia nigra, many GDNF-immunoreactive neurons were seen. This labeling represents retrograde transport of GDNF after injections of lenti-GDNF into the right striatum. Further, dense GDNF-immunoreactive fiber staining, representing anterograde transport of the trophic factor, was seen within the right substantia nigra pars reticulata. Tissue punches taken at the time of death revealed significant levels of GDNF produced by striatal cells 8 months after lenti-GDNF injections. On the side without a striatal injection, 0.130 ± 0.062 and 0.131 ± 0.060 ng/mg protein of GDNF were seen in the caudate nucleus and putamen, respectively. Significantly higher GDNF levels were observed within the caudate nucleus (2.25 ± 0.312 ng/mg protein;P < 0.001) and putamen (3.5 ± 0.582 ng/mg protein; P < 0.001) on the lenti-GDNF–injected side.

Our study demonstrates that delivery of GDNF cDNA into the nigrostriatal system using a lentiviral vector system can potently reverse the structural and functional effects of dopamine insufficiency in nonhuman primate models of aging and early Parkinson's disease. Most critically, lenti-GDNF delivery prevented the motor deficits that normally occur after MPTP administration. In this regard, functional disability was prevented on both a subjective clinical rating scale modeled after the Unified Parkinson's Disease Rating Scale and an objective operant motor test. Consistent expression of GDNF was observed in aged and lesioned monkeys with significant and biologically relevant levels of GDNF observed for up to 8 months after lentivirus injection. Indeed, the 2.5 to 3.5 ng/mg protein of GDNF produced after lenti-GDNF injections compares very favorably to the 50 to 152 pg/mg protein of striatal GDNF produced after intrastriatal adenovirus injections in monkeys (28).

This consistent gene expression occurred without significant toxicity to aged monkeys, and minor toxicity in two of the MPTP-treated monkeys, supporting our previous observations (15). Still, the death of two monkeys needs to be addressed. Pathological analyses revealed only a mild necrosis from multifocal random hepatocellular coagulation in these animals, and this was not deemed to be the cause of death. No other young adult or aged monkeys from this or a previous study (15) displayed morbidity or mortality after lentivirus injections. Further, detailed necropsies from the remaining MPTP-treated animals failed to reveal any relevant pathology. Although the absolute cause of death remains elusive, we hypothesize that the death of these two monkeys relates to the impact of the surgical procedure 1 week after the MPTP injections and is unrelated to the lentivirus injection.

In aged monkeys, lentiviral delivery of GDNF augmented host nigrostriatal function as determined by a variety of morphological, physiological, and neurochemical dependent measures. In this regard, lenti-GDNF increased the size and number of TH-immunoreactive neurons within the substantia nigra; increased the expression of TH mRNA within these neurons; increased the levels of dopamine, dopaminergic metabolites, and dopaminergic markers in the striatum; and increased FD uptake within the striatum as determined by PET scan. Enhanced nigrostriatal dopamine function was consistently associated with the expression of lentivirally delivered GDNF, as enhanced nigrostriatal function was only seen on the side with robust gene expression.

We used aged monkeys to model specific cellular changes that occur in aging and the earliest aspects of PD. Phenotypic down-regulation of the TH gene and protein are among the earliest pathological events seen within the substantia nigra in PD (17), and analogous changes are seen in aged rhesus monkeys (16). The number of TH-immunoreactive nigral neurons seen in lenti-βGal–injected animals was similar to that previously reported for aged rhesus monkeys (16). In contrast, lenti-GDNF–treated aged monkeys displayed nigral neurons in numbers similar to those seen in young adult animals. The possibility that the lenti-GDNF spurred neurogenesis of dopaminergic nigral neurons cannot be ruled out. However, the delivery of lenti-GDNF to the nigral region resulted in transgene expression throughout the midbrain. Yet, TH-immunoreactive neurons were observed only within established catecholaminergic nuclei and not in ectopic midbrain locations. A more parsimonious explanation is that GDNF up-regulated TH-immunoreactivity in aged nigral neurons that had previously down-regulated TH expression below detectable levels. The enhanced TH mRNA expression seen within nigral neurons after lenti-GDNF treatment supports this interpretation.

Lenti-GDNF also prevented the behavioral and neuroanatomical effects of MPTP-induced nigrostriatal degeneration. It is notable that, unlike many other neuroprotection paradigms, the lenti-GDNF injections were performed after the parkinsonian state was initiated, thus better modeling what can be attempted in PD patients. The exact mechanism by which lenti-GDNF exerted its effects requires further elucidation. It is clear that neuroprotection was achieved within the substantia nigra, as these neurons do not degenerate within a week of MPTP treatment (29). However, striatal fibers can degenerate during this time, and whether the GDNF is preventing degeneration or inducing sprouting of degenerating fibers still needs to be established. Indeed, there is evidence for both mechanisms as some animals displayed fiber morphology and topography indicative of regeneration.

A critical question is whether preservation of striatal innervation, nigral perikarya, or both, is required for functional recovery. Although the number of animals in our study is too small to provide a definitive answer, it is notable that all lenti-GDNF–treated monkeys had complete preservation of nigral perikarya. Yet, functional recovery on the hand-reach task was absent only in the one monkey with sparsest striatal reinnervation. Thus it appears that GDNF-mediated striatal reinnervation is critical for functional recovery in nonhuman primates, a concept supported by recent studies performed in rodents (30, 31). The failure to potently protect dopaminergic innervation in the one monkey may be due to variability in the speed by which nigrostriatal fibers are lost after MPTP. At the time of the lenti-GDNF injections, dopaminergic fibers in this monkey may have regressed to a level where access to the GDNF was limited, and regrowth to the striatum was impossible.

Not only was lenti-GDNF capable of preventing the degeneration of nigrostriatal neurons in MPTP-treated monkeys, it augmented many of the morphological parameters relative to the “intact” side. It is likely that the unilateral 3-mg MPTP dose induced a small loss of TH-immunoreactive neurons on the contralateral side. Thus the increased numbers of TH-immunoreactive neurons may reflect complete neuroprotection on the side of GDNF expression contrasted with a small loss of TH-immunoreactive neurons on the side not injected.

We injected lentivirus into both the striatum and substantia nigra in order to maximize the chance for an effect. For lenti-GDNF therapy to be a practical clinical approach, studies determining the regions of GDNF delivery critical to reverse progressive nigrostriatal degeneration are needed. The importance of related biological events such as anterograde transport of GDNF from injection sites to target regions also needs to be established. Finally, potential adverse events resulting from lenti-GDNF inducing supranormal levels of striatal dopamine needs to be evaluated. Toward this end, vectors with built-in inducible systems that can modulate gene expression in cases of dose-limiting side effects need to be developed. Still, the reversal of slowly progressive cellular phenotypic changes seen in aged monkeys, combined with the structural and functional neuroprotection and regeneration seen in MPTP-treated monkeys, indicates that lentiviral delivery of GDNF may provide potent clinical benefits for patients with PD.

  • * To whom correspondence should be addressed. E-mail: jkordowe{at}rush.edu

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