Macrophage-mediated GDNF delivery protects against dopaminergic neurodegeneration: a therapeutic strategy for Parkinson's disease

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinson's disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH(+) terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.

Figure 1

Lentiviral vector with MSP restricts transgene expression to macrophages. (a) Schematics of lentiviral vector expressing rat GDNF or GFP driven by MSP with a CD68 mini-promoter. (b) Peripheral blood flow cytometry analysis of MSP-GFP mice showing GFP expression mostly in CD11b⁺ cells (n = 10, P < 0.0001) 3 weeks after transplantation. (c) About 7% of the CD11b^‐ cells expressed very low levels of GFP (n = 10, P < 0.0001). (d,e) GDNF levels by enzyme-linked immunosorbent assay in the blood plasma (d, n = 5) and substantia nigra (e, n = 5, P < 0.002) of MSP-GFP and MSP-GDNF mice 17 weeks after transplantation. GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; MSP, macrophage-specific synthetic promoter.

Figure 2

Gene-modified macrophages are recruited in large numbers to substantia nigra following MPTP-induced neurodegeneration. (a) Midbrain sections of MSP-GFP mice treated with MPTP showing GFP⁺ cells expressing microglial marker Iba1 in the nigra. (b) Semiquantitative analysis of GFP⁺ cells in the substantia nigra of saline- and MPTP-treated MSP-GFP mice. Each bar represents the mean ± standard error of the total number of GFP⁺ cells per five representative sections of substantia nigra pars compacta per animal (n = 4, P < 0.0001). (c) Midbrain sections of MSP-GFP mice treated with saline and MPTP showing TH-immunostained neurons and GFP⁺ cells in the nigra. Note the large number of GFP⁺ cells in the nigra of MPTP-treated mice compared with control. The analysis was performed 9 weeks after MPTP treatment. GFP, green fluorescent protein; TH, tyrosine hydroxylase.

Figure 3

Macrophage-mediated GDNF delivery protects nigral dopaminergic neurons from MPTP-induced degeneration. (a) Midbrain sections of MSP-GFP and MSP-GDNF mice showing TH-immunostained cell bodies and their processes in the substantia nigra 3 weeks after MPTP treatment. Whereas MPTP treatment dramatically reduced the number of TH⁺ cell bodies and fibers in the substantia nigra pars compacta (SNpc) of MSP-GFP mice, a significant protective effect was observed in MSP-GDNF mice. Also note the sparing of TH⁺ processes running through the substantia nigra pars reticulata (SNpr). (b,c) Plots of quantitative stereologic data illustrating the protective effect of macrophage-mediated GDNF delivery at 3 weeks (b, ***P < 0.001) and 9 weeks (c, ***P < 0.001) after MPTP treatment. The number of animals used in each group is shown in parentheses. GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; MSP, macrophage-specific synthetic promoter; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; TH, tyrosine hydroxylase.

Figure 4

Macrophage-mediated GDNF delivery protects dopaminergic terminals in the striatum from MPTP-induced degeneration. (a) Coronal sections of forebrain showing immunostaining of TH⁺ terminals in the striatum of MSP-GFP and MSP-GDNF mice 3 weeks after MPTP treatment. In the MSP-GFP mice, after MPTP treatment, there is a dramatic reduction in the density of TH⁺ terminals, whereas a substantial portion of TH⁺ terminals is spared in the striatum of MSP-GDNF mice. (b,c) Plots of quantitative data illustrating a protective effect of macrophage-mediated GDNF delivery at 3 weeks (b, *P < 0.05) and 9 weeks (c, ***P < 0.001) after MPTP treatment as assessed by optical density measurement of TH⁺ terminals in the striatum. The number of animals used in each group is shown in parentheses. (d) TH⁺ terminals in the dorsolateral aspect of the striatum showing the regenerative response to macrophage-mediated GDNF delivery. Note many thick TH⁺ fibers with branches in the striatum of MPTP-treated MSP-GDNF mice. GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; MSP, macrophage-specific synthetic promoter; TH, tyrosine hydroxylase.

Figure 5

Macrophage-mediated GDNF delivery enhanced striatal levels of dopamine and its metabolites. HPLC analysis revealed a significantly higher level of (a) dopamine (n = 5, **P < 0.0275), and its metabolites (b) DOPAC (n = 5, ***P < 0.0037) and (c) HVA (n = 5, ***P < 0.0031) in the striatum of MPTP-treated MSP-GDNF mice, whereas nigral levels of (d) 5-HT (n = 5 in each group, P > 0.9394) and its metabolite (e) 5-HIAA (n = 5 in each group, P > 0.1898) did not differ significantly between MSP-GFP and MSP-GDNF mice after MPTP treatment. The analysis was performed 3 weeks after MPTP treatment.

Figure 6

Macrophage-mediated GDNF delivery improved functional recovery without major side effects. (a) Total activity assessed by open field test. Macrophage-mediated GDNF delivery reversed MPTP-induced hypoactivity (*P < 0.05). The number of animals used in each group is shown in parentheses, and the test was repeated four times. (b) MPTP treatment reduced 24-hour food intake normalized for weight in MSP-GFP mice, whereas food intake in MSP-GDNF mice was restored to that in saline-treated control mice (n = 5 and repeated three times, **P < 0.01). None of the treatment groups showed signs of allodynia, as assessed by the (c) frequency and (d) duration of the paw withdrawal response after application of acetone to the mid-plantar surface of the hindpaw (n = 4 and repeated three times). All the behavior tests represented in this figure were performed 8 weeks after MPTP treatment. (e) Mean change from initial body weight is expressed ± standard error. Although both the groups continued to gain additional weight, over time, MSP-GDNF mice gained significantly less weight than did MSP-GFP mice (P < 0.001; repeated-measures ANOVA). The number of animals in each group is shown in parentheses.