PMC

Short and long-term follow-up of numbers of nigral DA neurons and levels of striatal dopamine after unilateral neuronal or astrocytic GDNF expression. (a,c,e,g) Stereological assessment of nigral DA neuron numbers and (b,d,f,h) HPLC-determined striatal dopamine levels after unilateral striatal vector application of high-titre in a–d and low-titre in e–h. AAV-5-hSYN-EGFP, AAV-5-hGFAP-EGFP, AAV-5-hSYN-GDNF, and AAV-5-hGFAP-GDNF vectors for the ipsilateral (“inj”) and contralateral (“con”) hemisphere at 2 weeks in a,b,e,f and 3 months after MPTP lesion in c,d,g,h. Values given as mean ± SD, n = 5–6 mice for each vector type, vector dosage, and time point. DA, dopamine; EGFP, enhanced green fluorescent protein; HPLC, high-performance liquid chromatography; GDNF, glial cell line-derived neurotrophic factor.

Differences in delivery of GDNF to the nigra after neuronal versus astrocytic striatal expression. (a,c,e,g,l) GDNF immunoreactivity after unilateral striatal vector applications of high-titres of AAV-5-hSYN-GDNF or (b,d,f,h) AAV-5-hGFAP-GDNF, detected at 2 weeks in a,b,e,f,l and 3 months after MPTP treatment in c,d,g,h. (i,j) Absence of GDNF immunoreactivity after transduction with EGFP encoding vectors. Representative pictures from n = 4 mice per group. (m) Projections of striatal and/or pallidal neurons into the substantia nigra pars reticulata are demonstrated by fibrous EGFP fluorescence, (for more details see Supplementary Figure S3). (k) Nigral dopaminergic neurons are shown by TH immunoreactivity in (k), while GDNF immunoreactivity ventral to the pars compacta is shown in l. EGFP, enhanced green fluorescent protein; GDNF, glial cell line-derived neurotrophic factor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Schematical representation of GDNF delivery to ipsilateral and contralateral structures after expression in neurons versus astrocytes of the left mouse striatum. Injection of (a,b) high titre vectors and (c,d) low titre vectors. GDNF levels as determined by ELISA at 2 weeks after MPTP (for absolute values see Supplementary Table S1) are given within the respective structure (striatum, top row; nigra, bottom row), as n-fold GDNF level as compared to AAV-EGFP transduced controls. The percentage of striatal GDNF detected in the ipsilateral nigra is given besides the connecting arrows. Conditions *, ^#, **, ***, and **** demonstrated protection of DA neurons, fibres, and striatal DA at 2 weeks and 3 months after MPTP; condition ^### demonstrated restoration of striatal DA levels at 3 months after MPTP; conditions ^## and ^#### did not show any neuroprotection. N = 4–5 mice for each condition. DA, dopamine; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; GDNF, glial cell line-derived neurotrophic factor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Astroglial and neuronal targeting of AAV-5 vectors in the lesioned rodent parkinsonian striatum. (a) MPTP-induced striatal gliosis in mouse brain as demonstrated by GFAP immunohistochemistry (GFAP-IHC) is shown before lesion, (b) 2 weeks after lesion, and (c) 3 months after lesion. (d) EGFP expression by the AAV-5-hSYN-EGFP vector, (e) GFAP-IHC of the same section, and (f) absence of any astrocytic EGFP expression by the EGFP/GFAP overlay. (g) EGFP expression by the AAV-5-hGFAP-EGFP vector, (h) NeuN-IHC of the same section, and (i) absence of any neuronal transgene expression by EGFP/NeuN overlay. (j) Absence of transgene expression in microglia by EGFP/IBA1-IHC overlay is shown for the AAV-5-hSYN-EGFP vector, (k) for the AAV-5-hGFAP-EGFP vector for 2 weeks after MPTP and in (l) for 3 months after MPTP. (m) Stereological quantification of transgene-expressing astrocytes and neurons after AAV-5-hSYN-EGFP or AAV-5-hGFAP-EGFP transduction, gray bars showing astrocyte numbers × 10⁴/mm³ striatal tissue, black bars showing neuron numbers × 10⁴/mm³ striatal tissue. (n) Specific targeting of AAV-5-hGFAP-EGFP to astrocytes in the 6-OHDA lesioned rat brain as overlay of EGFP fluorescence with GFAP-IHC and (o) absence of neuronal transgene expression from this vector as overlay of EGFP fluorescence with NeuN-IHC. Representative pictures from n = 3 mice per group. Bar = 400 µm in a–c; 100 µm in d–o. ns, not significant. 6-OHDA, 6-hydroxy-dopamine; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; hGFAP, human glial fibrillary acidic protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Astrocyte-derived GDNF functionally restores motor performance and protects from DA neuron degeneration in the partial 6-OHDA lesion paradigm of the rat brain. (a) The time scale of the experiment. 6-OHDA = unilateral striatal injection of 6-hydroxy dopamine; APO = apomorphine application to induce rotation behaviour; AAV = unilateral striatal injection of AAV vectors; time given in weeks. (b) Rotations counted within 30 minutes ± SD at 1, 2, 7, and 17 weeks after 6-OHDA injections. Ctrl = PBS injected rats; AAV-5-hGFAP-GDNF = vector-injected rats without 6-OHDA lesion; 6-OHDA + AAV-EGFP = rats injected with AAV-5-GFAP-EGFP control virus after 6-OHDA lesion; 6-OHDA + AAV-GDNF = rats injected with the AAV-5-GFAP-GDNF vector after 6-OHDA lesion. (c) Weight gain of animal groups is exemplarily shown for 3 and 14 weeks after 6-OHDA lesion. (d) Immunohistochemical staining for tyrosine hydroxylase (TH). Upper panel: low maginification view of the ipsilateral and contralateral nigra of animals injected with AAV-5-hGFAP-EGFP or AAV-5-hGFAP-GDNF after 6-OHDA lesion. Middle panel: high magnification of the nigra ipsilaterally (left) and contralaterally (right) after 6-OHDA and AAV-5-hGFAP-EGFP injection. Lower panel: high magnification of the nigra ipsilaterally (left) and contralaterally (right) after 6-OHDA and AAV-5-hGFAP-GDNF injection. Arrows point to DA neurons with reduced TH immunoreactivity. (e) Stereological quantification of dopaminergic neurons. 6-OHDA, 6-hydroxy-dopamine; AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; GDNF, glial cell line-derived neurotrophic factor; hGFAP, human glial fibrillary acidic protein; PBS, phosphate-buffered saline.