Clinically relevant effects of convection-enhanced delivery of AAV2-GDNF on the dopaminergic nigrostriatal pathway in aged rhesus monkeys

Abstract

Growth factor therapy for Parkinson's disease offers the prospect of restoration of dopaminergic innervation and/or prevention of neurodegeneration. Safety and efficacy of an adeno-associated virus (AAV2) encoding human glial cell-derived neurotrophic factor (GDNF) was investigated in aged nonhuman primates. Positron emission tomography with 6-[(18)F]-fluoro-l-m-tyrosine (FMT-PET) in putamen was assessed 3 months before and after AAV2 infusion. In the right putamen, monkeys received either phosphate-buffered saline or low-dose (LD) or high-dose (HD) AAV2-GDNF. Monkeys that had received putaminal phosphate-buffered saline (PBS) infusions additionally received either PBS or HD AAV2-GDNF in the right substantia nigra (SN). The convection-enhanced delivery method used for infusion of AAV2-GDNF vector resulted in robust volume of GDNF distribution within the putamen. AAV2-GDNF increased FMT-PET uptake in the ipsilateral putamen as well as enhancing locomotor activity. Within the putamen and caudate, the HD gene transfer mediated intense GDNF fiber and extracellular immunoreactivity (IR). Retrograde and anterograde transport of GDNF to other brain regions was observed. AAV2-GDNF did not significantly affect dopamine in the ipsilateral putamen or caudate, but increased dopamine turnover in HD groups. HD putamen treatment increased the density of dopaminergic terminals in these regions. HD treatments, irrespective of the site of infusion, increased the number of nonpigmented TH-IR neurons in the SN. AAV2-GDNF gene transfer does not appear to elicit adverse effects, delivers therapeutic levels of GDNF within target brain areas, and enhances utilization of striatal dopamine and dopaminergic nigrostriatal innervation.

Figures

FIG. 1.

Effect of AAV2-GDNF on FMI uptake and locomotor activity in aged monkeys. (A) Percent change in FMT-PET signal over baseline (pre-infusion) 3 months after AAV2-GDNF infusion in the left (contralateral, gray) vs. right (ipsilateral, black) striatum. Inset: Representative PET images from an animal that received a low dose of AAV2-GDNF in putamen. The ipsilateral infusion site is on the right in each image. A trend toward increased FMT-PET is evident in all three groups regardless of site of infusion or dose. When all animals were pooled (lower left panel), 2-way ANOVA revealed a significant effect of AAV2-GDNF infusion (*p < 0.05). (B) Locomotor activity, measured electronically over a 12-h daytime period at baseline (gray) and 6-months after surgery (black). When all the AAV2-GDNF treatment groups were pooled and changes from baseline activity were calculated (lower right panel), locomotor activity in the control group declined (gray), whereas AAV2-GDNF-treated animals (black) tended to have increased activity (*p < 0.05).

FIG. 2.

Effect of infusion site and vector dose on immunohistochemical distribution of GDNF protein and nigral TH staining. (A) Infusion of 75 μl of AAV2-GDNF into monkey putamen produced extensive expression of GDNF in putamen. The circle represents the approximate diameter of a human putamen for comparison (10 mm). The numbers in the gray dots indicate the concentration of GDNF, expressed as nanograms per milligram of protein. This section is from a monkey that received a high putaminal dose of AAV2-GDNF. (B) Low-dose (LD) AAV2-GDNF to putamen. (C) High dose of vector delivered to the SN. Note the prominent caudal staining. (D–G) Effect of AAV2-GDNF on nigral TH staining. GDNF expression increased TH staining independent of site (putamen or SN). HD increased TH more than did LD. Black bar in panel 2C represents a 2-mm segment applicable to panels B–G. The circle in panel A represents a 10-mm diameter centered on the infusion site.

FIG. 3.

Effect of GDNF on dopamine and principal metabolites in the putamen. Open columns, control animals that received a PBS infusion; hatched columns, low-dose putamen group; gray columns, substantia nigra group; solid columns, high-dose putamen group. Statistical indicators are as follows: *p < 0.05, **p < 0.01, two-way ANOVA, followed by one-way ANOVA post-hoc tests, and Newman–Keuls post-hoc comparisons with PBS group.

FIG. 4.

Effect of GDNF on dopamine and metabolites in the caudate nucleus. Open columns, control animals that received a PBS infusion; hatched columns, low-dose putamen group; gray columns, substantia nigra group; solid columns, high-dose putamen group. Statistical indicators are as follows: *p < 0.05, two-way ANOVA, followed by one-way ANOVA post-hoc tests and Newman–Keuls post-hoc comparisons with PBS control.

FIG. 5.

Tyrosine hydroxylase immunoreactivity in the putamen after infusion of AAV2-GDNF to the putamen. Open columns, control animals that received a PBS infusion; hatched columns, low-dose putamen group; gray columns, substantia nigra group; solid columns, high-dose putamen group. Columns and error bars represent mean (±SEM) optical density measurements (relative to white matter). (A) Putamen: Two-way ANOVA revealed a significant interaction between hemisphere and treatment (p < 0.05); unpaired post-hoc test for right side: PBS versus high-dose putamen, p < 0.05; one-way ANOVA, p < 0.05. Post-hoc analyses by unpaired t tests indicated a significant difference between PBS and high-dose putamen treatment groups (*p < 0.05). In addition, mean optical density in the low-dose putamen group was significantly less than that measured in the high-dose putamen group (#p < 0.05). (B) Caudate nucleus: Two-way ANOVA revealed a significant effect of side (p = 0.02); one-way ANOVA (p < 0.01). Unpaired t tests (one-tailed): PBS versus HD putamen treatment (*p < 0.05), LD putamen versus HD putamen (#p < 0.01). (C) False color TH staining of representative sections from (i) LD putamen, (ii) SN, and (iii) HD putamen.

FIG. 6.

Effect of high-dose GDNF on nigral neurons. Sections from animals that received either PBS (open columns), a high dose of AAV2-GDNF in SN (gray columns), or an equivalent dose in putamen (solid columns) were subjected to stereology (see Materials and Methods). Cells were counted in 2 sections, 10 sections apart, at the level of the third nerve rootlets. (A) Total population of dopaminergic (TH) neurons. (B) Number of TH-containing nigral neurons after high-dose vector treatment. This subset of neurons does not contain neuromelanin deposits (TH-IR ONLY). (C) Vector treatment also increased cell body area (μm2) in nigral dopaminergic neurons containing both TH and neuromelanin or in neurons containing neuromelanin only (D). The cell body of neurons containing either TH only or Nissl neurons without TH or neuromelanin did not increase in size. Statistical analysis: *p < 0.05, two-way ANOVA, followed by one-way ANOVA post-hoc tests, and Newman–Keuls post-hoc comparisons with PBS control.

FIG. 7.

Composite figure of TH IR showing midbrain region containing SN and MFB (medial forebrain bundle). Shown is TH staining of a representative nonhuman primate that received high-dose AAV2-GDNF administration into the right putamen (A–C) compared with a control animal (D–F) that received a PBS infusion into the right putamen. (B) and (E) clearly show GDNF-induced upregulation of dopaminergic neurons in the SNc and sprouting of TH-IR fibers in the MFB in this GDNF-treated animal. Upregulation appears to be bilateral, probably because of cross-over of GDNF to the contralateral hemisphere in line with contralateral effects on DA levels and fiber density (Figs. 3 and 5). In contrast, the PBS-treated animal indicates the normal appearance of dopaminergic cells in the SNc and TH-IR fibers in the MFB. Scale bars: (B and E) 500 μm; (A, C, D, and F) 20 μm.