Anterograde axonal transport of AAV2-GDNF in rat basal ganglia

Abstract

We elucidated the effects of parkinsonian degeneration on trafficking of AAV2-GDNF in the nigro-striatum (nigro-ST) of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Vector infused into striatum (ST) was transported to substantia nigra (SN), both pars compacta (SNc), and pars reticulata (SNr). In the lesioned hemisphere, glial cell line-derived neurotrophic factor (GDNF) immunoreactivity was only found in SNr consistent with elimination of SNc dopaminergic (DA) neurons by 6-OHDA. Further analysis showed that striatal delivery of AAV2-GDNF resulted in GDNF expression in globus pallidus (GP), entopeduncular nucleus (EPN), and subthalamic nucleus (STN) in both lesioned and unlesioned hemispheres. Injection of vector into SN, covering both SNc and SNr, resulted in striatal expression of GDNF in the unlesioned hemisphere but not in the lesioned hemisphere. No expression was seen in GP or EPN. We conclude that adeno-associated virus serotype 2 (AAV2) is transported throughout the nigro-ST exclusively by anterograde transport. This transport phenomenon directs GDNF expression throughout the basal ganglia in regions that are adversely affected in Parkinson's disease (PD) in addition to SNc. Delivery of vector to SN, however, does not direct expression of GDNF in ST, EPN, or GP. On this basis, we believe that striatal delivery of AAV2-GDNF is the preferred course of action for trophic rescue of DA function.

Figure 1

Transport of glial cell line–derived neurotrophic factor (GDNF) throughout the nonlesioned and lesioned nigrostriatal pathway after injection of AAV2-GDNF to the striatum (ST). GDNF-immunostaining of the coronal sections after bilateral striatal injection of AAV2-GDNF into unilateral 6-hydroxydopamine lesioned rats at (a) 4 weeks and (b) 25 weeks after striatal AAV2-GDNF. GDNF staining was widely distributed throughout most of both hemispheres (top images) and in the substantia nigra (SN). GDNF staining in lesioned SN, however, was less prominent. Strong GDNF expression was seen within SNr in both hemispheres (c) high magnification, bottom images). On the lesioned side, little GDNF signal was seen in substantia nigra pars compacta (SNc). Approximate anatomical coordinates are + 2.0 anterior from bregma (ST level) and –5.20 posterior from bregma (SN).

Figure 2

Glial cell line–derived neurotrophic factor (GDNF), glutamic acid dehydrogenase (GAD) and tyrosine hydroxylase (TH) staining of substantia nigra (compacta and reticulata) after bilateral AAV2-GDNF injection to the striatum. (a) Brain slice schematic taken from the rat brain atlas corresponds to substantia nigra pars reticulata (SNr) and compacta (SNc), ventral tegmental area (VTA) and medial terminal nucleus (MT) - posterior to bregma: −5.20 mm. (b) GAD65/67-staining after color replacement (red to blue) with Adobe Photoshop. (c) GDNF and (d) TH staining of coronal sections without any color correction. Panel e depicts merging of b and c. Panel f depicts merging of b,c, and e. For details see Materials and Methods.

Figure 3

Distribution of glial cell line–derived neurotrophic factor (GDNF) after striatal AAV2-GDNF injection. (a) The external globus pallidus (GP) contained GDNF-positive cells (single arrow) and GDNF-positive fibers in both the unlesioned (left panels) and lesioned (right panels) hemisphere. (b) Strong intracellular (single arrow) and extracellular GDNF signal was also detected in entopeduncular nucleus (EPN) again in both hemispheres. (c) GDNF was also present in the subthalamic nucleus (STN) similar in both lesioned and unlesioned STN.

Figure 4

Transport of glial cell line–derived neurotrophic factor (GDNF) throughout the nonlesioned and lesioned nigrostriatal pathway after injection of AAV2-GDNF to the substantia nigra. (a) GDNF staining is widely distributed throughout infused nigral region in both unlesioned and lesioned hemisphere (bottom image). Striatal GDNF expression (diffuse pattern due to secretion of the protein) was seen in the unlesioned striatum but not in the lesioned striatum (top image). These data indicate that nigral injection of AAV2-GDNF does not result in striatal expression of GDNF. Approximate anatomical coordinates are + 2.0 anterior from bregma [striatum (ST) level] and –5.20 posterior from bregma [substantia nigra (SN) level]. (b) Note the asymmetrical tyrosine hydroxylase (TH) staining at the level of ST (top image) and SN (bottom image) after 6-hydroxydopamine lesion.