Neurospheres modified to produce glial cell line‐derived neurotrophic factor increase the survival of transplanted dopamine neurons

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival of dopamine neurons in a variety of in vitro and in vivo model systems. Therefore, it constitutes an important therapeutic protein with the potential to ameliorate dopamine neuronal degeneration in Parkinson's disease or to support dopamine neuronal replacement strategies. However, biophysical and practical considerations present obstacles for the direct delivery of the GDNF protein to CNS neurons. Here we show that rodent neural precursor cells isolated and expanded in culture as neurospheres (NS) can be genetically modified to express green fluorescent protein (GFP) or to release GDNF using lentiviral constructs. GDNF-NS increased the fibre outgrowth of primary embryonic dopamine neurons in cocultures, showing that the protein was released in biologically significant quantities. Furthermore, after transplantation into the 6-hydroxydopamine-lesioned rat striatum, GDNF-NS significantly increased the survival of cografted primary dopamine neurons. However, this was not reflected in behavioural recovery in these animals. We found that, by 6 weeks, few cells expressed GDNF or GFP, suggesting either that transgene expression was down-regulated over time or that the cells died. This may explain the initial effects on dopamine neuronal survival within the graft but the lack of long-term effect on subsequent fibre outgrowth and behaviour. Providing sustained levels of neural precursor-mediated transgene expression can be achieved following transplantation in the future; this approach may prove beneficial as an alternative therapeutic strategy in the cell-based management of Parkinson's disease.

Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

“…This confirms our earlier results using gene chip analysis for human neurospheres 19 and ELISA for rodent neurospheres. 27 Neurospheres co-infected with both lenti-tTA and the ind lenti-GDNF construct (hNPC-GDNF) produced large amounts of glycosylated GDNF revealed by Western blot analysis (Figure 1c, lane 3). This naturally glycosylated GDNF was similar to the glycosylated form produced by a non-human mammalian cell line (Figure 1c, lane 1) and was in contrast to the lighter-weight nonglycosylated human recombinant GDNF produced by bacteria ( Figure 1c, lane 2) and used for previous clinical trials.…”

“…26 Rodent neural progenitor cells that have been genetically modified to produce GDNF are physiologically active on dopamine neurons, both in vitro and following transplantation. 27 In addition, a mouse immortal cell line that produces GDNF, survives transplantation in rodent models of PD and prevents degeneration of dopamine neurons when transplanted before the lesion has been generated. 28,29 While these studies highlight the potential of GDNF-secreting cells, it will be crucial to demonstrate the efficiency of cells after rather than before the lesion to be relevant to PD.…”

Neurotrophic factors for the treatment of Parkinson's disease

“…Genetically-modified embryonic rat midbrain cells which over-express GDNF have been found to induce earlier functional recovery in 6-OHDA-lesioned rats than control grafts [68]. GDNF-overexpressing rat neural precursor cells also significantly increased the survival of co-grafted embryonic dopaminergic neurones [69]. Human neural progenitor cells have been used to deliver GDNF, which conferred protective effects on the lesioned nigrostriatal pathway in adult rats [70].…”

Gene therapy progress and prospects: Parkinson's disease

“…However, the delivery of potent biologically active peptides with short half-lives to the brain is difficult, and attempts at intraventricular infusion of recombinant GDNF were disappointing. The alternatives include continuous intraparenchymal infusion of recombinant GDNF, 3 transplantation of genetically modified cells as production sites for GDNF, [4][5][6][7][8] or vector-mediated transfer of the gene encoding GDNF into the CNS parenchyma.…”