We have previously reported expression of GDNF by means of AAV2-GDNF infusion by CED in the dopaminergic nigrostriatal pathway of aged NHP (Johnston et al.
), and demonstrated the potential clinical utility in MPTP-lesioned NHP (Eberling et al.
). In the present study, we evaluated safety issues regarding AAV2-GDNF administration to NHP brain. We demonstrate, in both models, an absence of tissue pathology with the exception of minor surgical trauma and a mild increase in anti-AAV2 capsid antibodies commonly seen in convective delivery procedures in NHP (Cunningham et al.
) and rodents (Sanftner et al.
). As shown in these earlier studies and in the present work, this type of infusion of AAV2 into striatum induces no cellular immune response, and this appears to be driven in part by the neuronal specificity of AAV2 in the brain (Bankiewicz et al.
). Little microglial/macrophage-mediated neuroinflammation in either putamen or substantia nigra was observed in the present study, except for that occurring in proximity to cannula tracks. Similarly, GFAP immunostaining did not show any strong correlation of GDNF upregulation with increased expression of GFAP-positive astrocyte in the putamen of all experimental subjects. In the aged primates, it seemed that the magnitude of GFAP immunoreactivity was greater in the GDNF treatment groups compared with the PBS controls. However, in the MPTP-lesioned primates, no detectable difference was observed between the GDNF treatment groups and PBS controls. It should be noted that MPTP lesioning before GDNF or PBS injection in these monkeys may confound the effect of GDNF on astrocytic activation (Chen et al.
). In the 14-month MPTP-lesioned primates, including both GDNF treatment and PBS control subjects, astrocytic activation appeared to resolve over time. Because there was no significant increase in proinflammatory cytokines/chemokines detected at the RNA level in the putamen of all treatment groups compared with the control group, increased GFAP+
astrocytes appear not to have conveyed any adverse effects.
The most important adverse finding was that nigral administration of AAV2-GDNF caused significant weight loss and increased GDNF levels in frontal cortical regions. This observation is in accordance with previous reports of weight loss in rats with nigral or hypothalamic delivery of AAV2-GDNF (Tumer et al.
; Manfredsson et al.
) and with reports describing similar, although transient, weight loss after intracerebroventricular (ICV) infusion of recombinant GDNF protein (Martin et al.
; Zhang et al.
). In our study, all the aged NHP received the same pattern of both nigral and putaminal injections of either AAV2-GDNF or PBS, indicating that surgery itself is unlikely to have caused weight loss in the SN group. The presence of viral vector itself (AAV2) in the nigra is also unlikely to be the cause of the weight loss; Manfredsson and colleagues showed that AAV2-GFP injected into the nigra as control had no effect. The SN group displayed reduced food intake for ~4 weeks after surgery, whereas most of the remaining NHP did not show decreased appetite, although a few animals showed brief (<1 week) postoperative loss of appetite, perhaps consistent with a prolonged postsurgical recovery period in aged NHP. Manfredsson and colleagues found a specific loss of adipose tissue, but not muscle, in GDNF-treated rats. They suggested a potential mechanism via stimulation by GDNF of a small population of corticotrophin-releasing factor (corticotrophin-releasing hormone [CRH]) neurons located in the medial parvocellular division (MPD) of the paraventricular nucleus of the hypothalamus. We have not, however, explicitly addressed this putative mechanism in our studies. Nevertheless, the fact that such significant weight loss can be induced by nigral GDNF expression is disturbing. Our findings, together with the work of others cited previously, suggest the need for considerable caution in proposing clinical studies in which GDNF or its homolog, neurturin, would be specifically directed to the substantia nigra. Such clinical protocols should be well supported by direct experiments with the vector system in question specifically designed to rule out weight loss as an adverse event.
Interestingly, putaminal GDNF does not cause weight loss either in intact or lesioned NHP, even though we have demonstrated considerable transport of GDNF from putamen to substantia nigra. We suspect that anterograde transport of AAV2-GDNF (and probably GDNF protein as well) to other areas of the midbrain are responsible. In lesioned animals, where the dopaminergic projections to the striatum have been substantially eliminated, transport of vector/gene product to the nigra is comparable to that seen in unlesioned animals. This suggests that anterograde, rather than retrograde, transport is the predominant means by which GDNF is disseminated to distal locations, although clearly retrograde transport of AAV2 also occurs under some conditions (Kaspar et al.
; Kells et al.
Our finding of TH-positive profiles in the MPTP-lesioned primates is interesting. This is the first report, to our knowledge, of the presence of TH-positive profiles in MPTP primates after GDNF administration. These enhanced TH-positive profiles, more evident in mildly lesioned hemispheres, were increased in those monkeys that received GDNF. It is possible that such profiles represent cross-sections of enlarged TH-positive fibers as previously reported by Suwelack and colleagues (2004
) in 6-OHDA-lesioned rats treated with a recombinant adenovirus containing GDNF. The fact that profiles are more abundant in the mildly lesioned hemisphere and in the ventral striatum and nucleus accumbens (partially affected by MPTP on the ipsilateral side) suggests that TH-positive profiles are in fact enlarged dopaminergic fibers. Whether these TH-positive profiles or “hypertrophic fibers” convey any beneficial or detrimental effect remains to be investigated. However, they did not appear to elicit any inflammatory responses, because no activated microglia surrounding these fibers were detected.
Taken together, the present findings demonstrated that putaminal administration of AAV2-GDNF at the indicated doses in aged and MPTP-lesioned NHP induced no significant immunological responses and no observed adverse side effects. Our findings support putaminal, but not nigral, AAV2-GDNF gene therapy as a safe therapeutic strategy for Parkinson's disease. This work raises substantial safety concerns for any proposed clinical studies in which nigral, in contrast to putaminal, delivery of GDNF or similar transforming growth factor-β family growth factors are contemplated.