Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats - PubMed (original) (raw)

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats

R J Mandel et al. Proc Natl Acad Sci U S A. 1997.

Abstract

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson's disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson's disease.

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Figures

Figure 4

Figure 4

Photomicrographs of nigral β-galactosidase expression 7 weeks after rAAV-CMV-lacZ injection. The nigral sections shown were taken from an rAAV-CMV-_lacZ_-injected rat that showed no evidence of a 6-OHDA lesion, and the rat was removed from the study. rAAV-CMV-_lacZ_-injected animals that were included in the study were found to have little or no β-galactosidase staining, probably because of the destruction of transduced cells by the subsequent striatal 6-OHDA lesion. (A) Low-power micrograph of the SN. The arrow indicates the injection site. (Bar = 200 μm.) (B) Higher-power micrograph of the same field. The arrow indicates the injection site. As has been observed (24, 41) most of the cells expressing β-galactosidase appear to be neurons. (Bar = 100 μm.)

Figure 1

Figure 1

Production of GDNF in vitro from rAAV-MD-GDNF-transduced HeLa cells. Released GDNF levels were measured after addition of increasing amounts of rAAV-MD-GDNF vector to cultures containing 3 × 105 HeLa cells. The GDNF was measured by ELISA from samples taken after 20 hr from the culture medium.

Figure 2

Figure 2

Nigral cell survival 4 weeks after a striatal 6-OHDA lesion. (A) Cell survival as estimated from cell counts of nigral FG+ cells. Nigral injection of rAAV-MD-rGDNF (solid bar, n = 6) resulted in a significantly greater number of FG+ cells surviving (indicated by *) after the striatal 6-OHDA lesion as compared with the rAAV-CMV-lacZ [open bar, n = 7, F(1, 11) = 20.1, P < 0.001]. (B) Cell survival estimated from cell counts of nigral TH+ cells. Nigral injection of rAAV-MD-rGDNF (solid bar, n = 6) resulted in a significantly greater number of TH+ cells surviving (indicated by *) after the striatal 6-OHDA lesion as compared with the rAAV-CMV-lacZ [open bar, n = 7, F(1, 11) = 6.9, P = 0.02]. Error bars represent +1 SEM.

Figure 3

Figure 3

Photomicrographs of intact and lesioned/vector-injected SN 4 weeks after a striatal 6-OHDA lesion. (A and C) Photomicrographs of TH immunocytochemical staining of intact SN from a representative rAAV-CMV-_lacZ_-injected animal and a representative rAAV-MD-rGDNF-injected rat, respectively. (B and D) Photomicrographs of TH staining of the SN from lesioned and vector-injected hemisphere of representative rAAV-CMV-_lacZ_-injected animal and a representative rAAV-MD-rGDNF-injected rat, respectively. The arrows indicate a vector injection site. The cell loss due to the 6-OHDA lesion seen in B was significantly diminished (see Results) by pretreatment with rAAV-MD-rGDNF as demonstrated in D. The bar in A = 200 μm and applies to A–D. (E and G) Photomicrographs of FG back-labeled SN from the intact hemisphere from a representative rAAV-CMV-_lacZ_-injected animal and a representative rAAV-MD-rGDNF-injected rat, respectively. (F and H) Photomicrographs of FG back-labeled SN from the lesioned and vector-injected hemisphere from a representative rAAV-CMV-_lacZ_-injected animal and a representative rAAV-MD-rGDNF-injected rat, respectively. In addition to the 6-OHDA-induced cell loss demonstrated in F, there are numerous shrunken cells, punctae, and fibers with varicosities that are not apparent in the rAAV-MD-rGDNF-injected nigra shown in H. This pattern of 6-OHDA-induced damaged in FG back-labeled SN has been reported previously (17, 21, 22). The bar in E = 100 μm and applies to E–H.

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