Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

Abstract

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson's disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson's disease.

Figure 1

Schematic representation of genomic HSV vectors. Both the control vectors THZ.1 and TSZ.1 and the Bcl2-expressing vector THZ/S-bcl2 are deleted for the immediate early genes ICP4 (both copies), ICP27, and ICP22, indicated by solid bars in the genome.

Figure 2

Bcl-2 expression in vitro. Western blot of lysates of infected Hig-82 cells shows a 26-kDa BCl-2 immunoreactive band in THZ/S-bcl2 but not mock- or THZ.1-infected cells.

Figure 3

Expression of Bcl-2 in cortical neurons in vitro inhibits naturally occurring cell death. (A) TUNEL staining of uninfected primary cortical neuron cultures at 3 weeks. The majority of residual shrunken nuclei in uninfected cultures was TUNEL-positive, whereas viable neurons were unlabeled. Similar patterns were seen in cultures at 2 and 4 weeks after plating. (B) Three weeks after infection, cultures infected with THZ/S-bcl2 demonstrated increased cell survival compared with uninfected cultures or cultures infected with THZ.1. Cell viability was determined by Alamar blue fluorescence, and the data are presented as a percentage of mock-infected cells. The data presented were obtained from three independent repetitions of the same experiment, with four wells in each group for each experiment. (P < .05 for the comparison of THZ.1 with THZ/S-bcl2 by t test.)

Figure 4

Transgene-driven expression of human Bcl-2 RNA in rat SN. RT-PCR from a single 40-μm section of rat SN demonstrates bcl-2 in vector-injected (S1–S3) but not control (C1–C2) brains. C3 is a water blank.

Figure 5

Expression of Bcl-2 in SN protects neurons from 6-OHDA toxicity. (A) Cell survival as estimated from cell counts of nigral FG⁺ cells 2 weeks after 6-OHDA lesion. THZ/S-bcl2 transfection resulted in a significantly greater number of FG⁺ cells surviving the lesion as compared with THZ.1 control transfected SN (n = 9 animals in each group, P < .05). (B) Cell survival as estimated from counts of TH-immunoreactive cells 2 weeks after 6-OHDA lesion. THZ/S-bcl2 transfection resulted in a significantly greater number of TH-immunoreactive cells surviving the lesion as compared with TSZ.1 control transfected SN (n = 9 animals in each group, P < .05). The experiment was repeated twice with the same results.

Figure 6

Photomicrographs of intact and lesioned/vector-injected SN 2 weeks after 6-OHDA lesion (FG⁺ cells). Representative photomicrographs of FG⁺ cells in contralateral intact SN (A and C) and ipsilateral SN injected with THZ/S-bcl2 (B) and TSZ.1 (D) are shown.

Figure 7

Photomicrographs of intact and lesioned/vector-injected SN 2 weeks after 6 OHDA lesion (TH-immunoreactive cells). Representative photomicrographs of TH-immunoreactive cells in contralateral intact SN (A and C) and from ipsilateral SN injected with THZ/S-bcl2 (B) and TSZ.1 (D).