Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease

Abstract

Cerebral dopamine neurotrophic factor (CDNF) protein has been shown to protect the nigrostriatal dopaminergic pathway when given as intrastriatal infusions in rat and mouse models of Parkinson's disease (PD). In this study, we assessed the neuroprotective effect of CDNF delivered with a recombinant adeno-associated viral (AAV) serotype 2 vector in a rat 6-hydroxydopamine (6-OHDA) model of PD. AAV2 vectors encoding CDNF, glial cell line-derived neurotrophic factor (GDNF), or green fluorescent protein were injected into the rat striatum. Protein expression analysis showed that our AAV2 vector efficiently delivered the neurotrophic factor genes into the brain and gave rise to a long-lasting expression of the proteins. Two weeks after AAV2 vector injection, 6-OHDA was injected into the rat striatum, creating a progressive degeneration of the nigrostriatal dopaminergic system. Treatment with AAV2-CDNF resulted in a marked decrease in amphetamine-induced ipsilateral rotations while it provided only partial protection of tyrosine hydroxylase (TH)-immunoreactive cells in the rat substantia nigra pars compacta and TH-reactive fibers in the striatum. Results from this study provide additional evidence that CDNF can be considered a potential treatment of Parkinson's disease.

Keywords: 6-OHDA; AAV; CDNF; GDNF; gene therapy.

Figure 1

Schematic drawing of the pAAV2-CDNF vector (A) and experimental design for evaluating the neuroprotective effect of AAV2-CDNF in a 6-OHDA partial lesion model of PD in rats (B).

Figure 2

The level of hCDNF protein in the rat striatum (A, B) and substantia nigra (SN) (D) following injection of AAV2-CDNF into the striatum measured by our CDNF-ELISA assay (n = 4/measure point). The protein expression was dependent on the injected AAV2 vector titer, when measured 4 weeks after delivery of the hCDNF gene (A), remained stable until the end of the experiment (12 weeks) (B), and was comparable to that of GDNF following AAV-GDNF injection (C; n = 3). CDNF protein could also be detected in the ipsilateral SN following intrastriatal AAV2-CDNF injection (D). *P < 0.05 versus AAV-CDNF 4 × 10⁷ and 2 × 10⁸ vg, one-way ANOVA and Games–Howell post hoc test, n = 4/group (A, B, D), n = 3 (C).

Figure 3

Expression of CDNF and GDNF proteins in the lesioned rat brain 12 weeks after injection of AAV2-CDNF or AAV2-GDNF into the striatum. Expression of CDNF (A) and GDNF (F) was detected around the injection tract and the expression of CDNF colocalized with the neuronal marker NeuN in the striatum (K). While CDNF protein was mainly found intracellularly, GDNF was evenly distributed in the tissue surrounding the injection tract (insets in A, F). Solitary CDNF-immunoreactive cells were detected in the ipsilateral globus pallidus (GP) (B) and substantia nigra (SN) (D). Some of the cells showing expression of hCDNF protein in the SNpc were TH positive (L). Following intrastriatal injection of AAV2-GDNF, GDNF signal could be detected in the GP (G) and in both SN pars compacta and SN pars reticulata (I). Background staining of CDNF and GDNF is shown in pictures C and E, and H and J, respectively. Scale bars: 200 μm (insets in A, F), 500 μm (B, C, D, E, G, H, I, J), and 10 μm (K, L).

Figure 4

Amphetamine-induced (2.5 mg/kg, i.p.) rotational behavior was measured for 120 min at 2, 4, 6, 8, and 10 weeks following the unilateral 6-OHDA lesion. Intrastriatal injection of AAV2-CDNF or AAV2-GDNF was able to attenuate the ipsilateral turning behavior. Intergroup comparisons were assessed using one-way ANOVA (results showed in the upper right corner of each graph) and Games–Howell post hoc test. *P < 0.05, **P < 0.01 versus control group, n = 9-18.

Figure 5

Tyrosine hydroxylase (TH) immunoreactivity in the rat striatum (A and B) and substantia nigra pars compacta (SNpc) (C, D, and E) 10 weeks post lesion (12 weeks after virus vector injection). Quantified results (A, C, and D) are given as percentage of the lesioned side as compared with the intact side. The white line across the dorsal striatum shows the site of optical density measurements (B). Treatment with AAV2-GDNF increased TH-reactive fiber density in the striatum as compared with the control group (A). Representative sections of TH staining in the striatum are shown in (B). Stereological analysis of nearly the entire SNpc (from 4.5 to 6.0 mm posterior to bregma) showed that none of the treatments were able to provide statistically significant protection of the TH-reactive neurons in the SNpc (C). AAV2-CDNF 1 × 10⁹ vg was able to protect the nigral TH-reactive cells in the central SNpc (D). Representative sections showing TH-reactive cells in the central SNpc are visualized in (E). *P < 0.05, **P < 0.01 versus control, one-way ANOVA, and Tukey HSD post hoc test, n = 9-18. Scale bar: 500 μm.

Figure 6

TH immunoreactivity in the striatum (STR) (A), globus pallidus (GP) (B), and substantia nigra pars reticulata (SNpr) (C) of the intact brain and 6-OHDA-lesioned rats treated with AAV2-GFP, AAV2-GDNF, or AAV2-CDNF. Twelve weeks post lesion, the 6-OHDA injections had caused an almost complete loss of TH-reactivity in the striatum, GP, and SNpr (AAV-GFP; A, B, and C). Injection of AAV2-GDNF 2 weeks before the 6-OHDA injections caused an increase of TH-positive fibers in the striatum, GP, and SNpr (AAV2-GDNF; A, B, and C). Treatment with AAV2-CDNF did not result in any clear sprouting, but caused an overall, partial protection of the TH-reactive fibers (A, B, and C) that was confirmed by the optical density measurements in the striatum (Fig. 5C). Compared with AAV2-CDNF, infusion of CDNF protein resulted in a denser meshwork of TH-reactive fibers close to the infusion tract (arrow) in the 6-OHDA-lesioned rats (D). Scale bars: 500 μm (A, B, C), 200 μm (D).