Regeneration of the MPTP-lesioned dopaminergic system after convection-enhanced delivery of AAV2-GDNF

Abstract

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinson's disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3-6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.

Figure 1.

Effect of AAV2-GDNF infusion on parkinsonian symptoms and FMT-PET. A, CRS scores in MPTP-lesioned macaques treated with AAV2-GDNF (black triangles) were significantly reduced compared with the PBS controls (gray circles). Two-way ANOVA statistics for the effect of treatment are shown for the three different survival periods. B, CRS scores after l-dopa administration (symbols and solid lines) were lower than the OFF l-dopa scores (dashed lines) for both AAV2-GDNF and PBS controls. C, Enhanced dopaminergic activity in the putamen after AAV2-GDNF delivery. FMT-PET was considerably increased bilaterally in AAV2-GDNF-treated macaques (light and dark gray bars) compared with the PBS controls. Twenty-two months after AAV2-GDNF delivery, the PET signal was further increased with the severely lesioned putamen showing a much greater percentage increase in FMT uptake than the moderately lesioned putamen. Bars are mean ± SEM. D, E, Direct correlations between the increase in FMT-PET and improvements in CRS were observed for both the moderately lesioned left (D) and severely lesioned right (E) putamen. These correlations were evident 6 months after treatment (open triangles) and were undiminished at 22 months (closed triangles). Dashed lines connect the 6- and 22-month measurements for the same macaques. Figure updated and modified from our preliminary report (Eberling et al., 2009).

Figure 2.

Dopamine and metabolites in severely and moderately lesioned putamen. A–D, HPLC assay of dopamine (DA) levels (A, B) and HVA:dopamine ratios (C, D) in the putamen of AAV2-GDNF and PBS control-treated macaques at 6-, 14-, and 24-month endpoints. E–H, Correlation analysis of HVA versus dopamine (E, F) and the rate of dopamine turnover (HVA:dopamine ratio) versus dopamine (G, H). Previously reported data (Oiwa et al., 2003) from naive and parkinsonian macaques are included for comparison [white bars in B and D, labeled points (+) in E–H]. **p < 0.01, two-tailed unpaired t test compared to PBS controls.

Figure 3.

Expression of GDNF within the basal ganglia. A, Bilateral GDNF expression in the putamen at 1, 6, 14, and 24 months (m) after AAV2-GDNF treatment. B, GDNF expression was closely correlated with enhanced TH staining for adjacent sections from an AAV2-GDNF-treated macaque. TH staining in a 14-month post-treatment PBS control animal is shown for comparison. The severe MPTP lesioning of the right hemisphere is evident in the PBS controls by the substantial lack of TH staining relative to the moderately lesioned left hemisphere. Restricted GDNF expression within the left putamen of this particular animal was clearly correlated with increased TH within the same region of the putamen (thick arrows). A parallel correlation was less robust within the severely lesioned right putamen where enhanced TH was most evident in the medial and ventral aspects of the putamen. Bilaterally enhanced TH expression within the globus pallidus was evident in AAV2-GDNF-treated animals (thin arrows) and correlated with the presence of GDNF. Anterograde transportation of GDNF to the subthalamic nucleus (T'd arrows) and substantia nigra (arrowheads) was most evident in the severely lesioned right hemisphere of this animal, consistent with the greater distribution of GDNF within the right putamen. Higher magnification images of GDNF expression are available in supplemental Fig. S1, available at www.jneurosci.org as supplemental material. Scale bar, 10 mm.

Figure 4.

TH-positive fibers and neurons in the putamen and substantia nigra. A–D, AAV2-GDNF-treated macaques had more extensive TH expression within the caudate–putamen (A) and mid-brain (B) than PBS controls (C, D). Severe MPTP-induced lesioning of the right hemisphere was evident by an almost complete lack of TH-positive fibers in the caudate–putamen; however, AAV2-GDNF treatment increased both the density and the size of TH-positive fibers. The extensive TH-positive fiber network in the left caudate–putamen prevented visualization of individual fibers but showed enhanced TH expression after AAV2-GDNF treatment and also the presence of TH-positive profiles (A, arrow) (additional images in supplemental Fig. S3, available at www.jneurosci.org as supplemental material). Bilateral upregulation of TH expression after AAV2-GDNF treatment was also evident in the substantia nigra where the asymmetry of the lesioning was evident by the number of surviving TH-positive neurons. While AAV2-GDNF-treatment increased TH expression, it was not evident whether there was an increase in the number of TH-positive neurons within the substantia nigra. Scale bars: low-magnification images, 5 mm; medium-magnification images, 200 μm; high-magnification images, 50 μm.

Figure 5.

Restoration of dopaminergic fibers within the putamen. A, B, Tyrosine hydroxylase staining intensity in the moderately lesioned left (A) and severely lesioned right (B) putamen of AAV2-GDNF-treated (gray bars) and PBS-treated (white bars) macaques. The mean intensity of TH staining measured in four different regions of the putamen is shown. TH staining intensity (mean ± SEM) is relative to the intensity measured in TH-negative cortex and white matter tracts in the same sections. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t tests between treatment groups. C, D, Representative TH staining of paraffin sections from PBS- and AAV2-GDNF-treated macaques. cc, Corpus callosum; Cd, caudate nucleus; DL, dorsal lateral putamen; C, central putamen; DM, dorsal medial putamen; Put, putamen; V, ventral putamen. Scale bar, 5 mm.