Our study of the time-course of GDNF overexpression in normal monkeys revealed that there was no decrease in expression between 1 and 6 months following injection of AAV2-GDNF in the caudate nucleus of monkeys. This outcome was in line with our expectations, based on the absence of toxicity, low immunogenicity and sustained transgene expression of AAV vectors using the chicken β-actin/cytomegalovirus (Tenenbaum et al., 2004
). These results showing sustained overexpression of GDNF in normal monkeys striatum justified an investigation of the potential of AAV2-GDNF to enhance the survival and outgrowth of grafted fetal DA neurons in MPTP-treated monkeys in a relatively long-term study. In fact, the volume of striatum occupied by transduced cells in the monkey was larger than predicted by rodent studies, in which rAAV serotype 2 vectors have delivered genes only to a restricted region surrounding the injection needle (Paterna et al., 2004
; Taymans et al., 2007
). While this apparent difference in distribution of GDNF may be due to species or methodological differences, the observation of substantial spread of GDNF promised adequate exposure of the graft to GDNF. Intrastriatal injection AAV2-GDNF was observed to increase markedly the survival and outgrowth of co-grafted fetal TH-positive cells in the striatum of MPTP-treated monkeys when examined 6 months after implantation. Thus, these data suggest that supplementing intrastriatal fetal VM grafts with in vivo injections of viral vector delivering the GDNF gene may decrease the number of VM grafts necessary to re-supply the host striatum with adequate levels of DA. This would be a significant achievement, as the use of eight donors for one human host (Hauser et al., 1999
; Olanow et al., 2003
) is an obstacle to neural grafting as a treatment option for Parkinson’s disease.
In addition to striatal cells expressing GDNF, we noticed other apparently non-cellular structures that stained for GDNF. These appeared in the vicinity and beyond the area of diffuse GDNF staining. Their appearance was not dependent on the presence of fetal tissue, as they appeared in brains injected with AAV2-GDNF vector alone, and in brains from our studies injected with other vectors carrying the GDNF gene, specifically AAV5 or equine infectious anemia virus, but not in brains injected with AAV2 vector harboring the gene for green fluorescent protein. In the time course study () their occurrence was more prevalent at later time points than at one month. While we do not have a definitive identification of these structures, it is possible that they are associated with extracellular matrix complexes (Lapchak et al., 1998
; Ruoslahti, 1996
; Venstrom and Reichardt, 1993
There have been indications from rodent experiments that overexpression of GDNF using a recombinant lentiviral vector can lead eventually to down-regulation of TH (see Introduction and Georgievska et al., 2004a
). While we do not have a time-course to suggest whether the level of TH expression is altered over the course of 6 months studied, our data showing a robust increase in TH+ cell survival and TH+ fiber outgrowth suggests that a down-regulation did not occur in our study in monkeys using a recombinant AAV. There are several plausible reasons for a more persistent beneficial effect of GDNF on TH in grafted fetal DA neurons in the monkey compared to the rat. As the development of primate brain is extended over a longer period of time than in rodents, it may be that TH in grafted DA neurons will be down-regulated in the monkey, if exposed to elevated GDNF levels for longer than the 6 months studied, possibly when the grafted neurons have reached a stage of maturity when they no longer would normally require high levels of GDNF. Alternatively, since the relationship between GDNF concentration, time of GDNF exposure and TH activity has not been defined, it may be that down-regulation of the enzyme will not occur over time if exposure to GDNF does not exceed a certain threshold. In addition, as TH in primate brain exists in multiple isoforms (Haycock, 2002
) which may be differentially regulated (Lehmann et al., 2006
), it is feasible that the effect of high GDNF concentrations over time will be different in the primate and rodent brain. Finally, the different parkinsonian models used here and in the rodent study (Georgievska et al., 2004a
) may have a bearing on the effects of GDNF on TH. So, while no evidence of TH down-regulation was seen in the current study, further work will be necessary to determine whether, and under what conditions, this may occur in primate brain.
Some parkinsonian patients that received fetal VM grafts in recent clinical trials developed off-medication dyskinesias or graft-induced dyskinesias (GID) (Freed et al., 2001
; Hagell et al., 2002
; Olanow et al., 2003
). The proportion of patients in these 3 studies with GID severe enough to constitute clinical therapeutic problems has been estimated to be between 7 and 15% (Hagell and Cenci, 2005
). There is yet no agreement on the cause(s) of this side-effect. Possible explanations have been based on the cellular composition of the graft, the immune response, and the integration of graft and host cells, and damage from the transplantation procedure (Hagell and Cenci, 2005
). A leading theory is that GID are due to an incomplete and uneven striatal DA reinnervation by fetal grafts (Carlsson et al., 2006
; Hagell and Cenci, 2005
; Steece-Collier et al., 2003
). If this latter suggestion is correct, then targeted overexpression of GDNF in combination with fetal grafts in the striatum in Parkinson’s disease may lessen the propensity for GID, as this treatment was observed to increase cell survival and outgrowth of donor DA neurons in the MPTP-treated monkey. While a possible differential effect of GDNF on outgrowth of A9 and A10 neurons needs to be investigated in vivo (Borgal et al., 2007
), the observed enhanced survival of grafted DA neurons by GDNF indicates that this combination strategy would enable fewer VM donors to be used to re-supply the parkinsonian striatum with adequate DA levels. The significance of reducing the number of donors required for one host brain in the transplantation treatment for Parkinson’s disease is that it would lessen both the trauma of the surgery and the risk of a deleterious immune response (Hagell and Cenci, 2005
). Thus, there are several theoretical arguments that favor the use of fetal VM grafts together with a viral vector carrying the GDNF gene to lessen the susceptibility for development of GID in Parkinson’s disease.
Another advantage to the use of viral vector delivered GDNF to support fetal grafts in Parkinson’s disease is that GDNF by itself may provide protection of the ongoing degenerating host DA neurons. In fact, clinical trials have been conducted to examine the effect of brain infusion of GDNF on clinical outcome in patients with Parkinson’s disease. While some encouraging data emerged, side-effects were noted, which may have resulted from the high concentrations of GDNF delivered locally (Eslamboli, 2005
; Kirik et al., 2004
; Sherer et al., 2006
). The use of viral vectors to deliver GDNF in a stable, and potentially a regulated manner, offers a more promising approach to protect remaining DA neurons in Parkinson’s disease than direct infusions of GDNF (Kordower, 2003
; McBride and Kordower, 2002
). Thus, in patients with Parkinson’s disease who have received fetal VM grafts, viral vector-mediated over-expression of GDNF might be expected to enhance the benefit of the transplant, in addition to retarding the neurodegeneration of the host nigrostriatal DA system.
In summary, the present studies have demonstrated a prolonged overexpression of GDNF in monkey brain following intrastriatal injection of an AAV2-GDNF, and that co-implantation of this viral vector in the vicinity of an intrastriatal fetal VM graft in the MPTP-treated monkey led to the increased survival and outgrowth of TH+ neurons. The use of viral vectors to overexpress GDNF, or other neurotrophic or neurotropic factors, in localized regions of the brain may allow neural grafting to become a viable treatment strategy for Parkinson’s disease. The beneficial effects of GDNF delivered by a viral vector reported here for fetal DA neurons also could be applied to tailor the fate of stem cells transplanted to the parkinsonian brain.