Neuroprotection by transgenic expression of glucose-6-phosphate dehydrogenase in dopaminergic nigrostriatal neurons of mice

Abstract

Oxidative damage to dopaminergic nigrostriatal (DNS) neurons plays a central role in the pathogenesis of Parkinson's disease (PD). Glucose-6-phosphate dehydrogenase (G6PD) is a key cytoprotective enzyme that provides NADPH, the major source of the reducing equivalents of a cell. Mutations of this enzyme are the most common enzymopathies worldwide. We have studied in vivo the role of G6PD overexpressed specifically in the DNS pathway and show that the increase of G6PD activity in the soma and axon terminals of DNS neurons, separately from other neurons or glial cells, protects them from parkinsonism. Analysis of DNS neurons by histological, neurochemical, and functional methods showed that even a moderate increase of G6PD activity rendered transgenic mice more resistant than control littermates to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotective action of G6PD was also observed in aged animals despite that they had a greater susceptibility to MPTP. Therefore, overexpression of G6PD in dopaminergic neurons or pharmacological activation of the native enzyme should be considered as potential therapeutic strategies to PD.

Figure 1.

Generation of pTH-G6PD transgenic mice. A, Scheme of the pTH-G6PD construct used to create the pTH-G6PD transgenic mouse. B, Representative agarose gel of PCR products of genomic DNA from some of the offspring produced by a founder mouse. The expected size of the PCR product was 330 bp. Lanes with sample #100 and #101, wild-type littermates; lanes with sample #189 and #190, G6PD transgenic mice; lane 5, a wild-type mouse; lane 6, water. C, Analysis of the G6PD mRNA level by in situ hybridization in the ventral mesencephalon and the adrenal gland (insets) of a wild-type (left) or a transgenic mouse (center). In situ hybridization to detect TH mRNA in ventral mesencephalon and adrenal gland (right) is shown for comparison.

Figure 2.

Determination of G6PD, TH, and DAT protein levels in the nigrostriatal pathway of pTH-G6PD transgenic mice. A, Immunocytochemical study indicating the lack of G6PD signal in wild-type animals (left) either in SN (top) or in striatum (St; bottom). A strong immunostaining was observed in the substantia nigra and the ventral tegmental area of transgenic mice (center, top). At large magnification, a marked signal for G6PD protein was seen in some striatal fibers of transgenic animals. As control, immunostaining against TH was performed (right). B, Protein from striatum was immunoblotted against TH (left, top), DAT (right, top), or tubulin (for normalization). Quantification of TH and DAT density in transgenic (Tg) mice was calculated as percentage of wild-type (Wt) animals (bottom panels); n = 8 for each group used in TH and DAT quantification. Statistical analysis was done using Student's t test. Tub, α-Tubulin. Error bars represent SEM.

Figure 3.

Specific expression of G6PD in DNS neurons. Immunofluorescence staining of a slice of ventral mesencephalon using antibodies against G6PD (red, A) and TH (green, B). Superposition of the two fluorescent signals is evidenced by the yellow color in the merged image in C. The insets show a region in the substantia nigra at large magnification. A similar experiment using antibodies against G6PD (red, D) and GFAP (green, E). The lack of superposition of the two fluorescent signals (merged image in F) indicates that G6PD was not expressed in glial cells.

Figure 4.

Protection against MPTP-induced toxicity by G6PD overexpression in young animals. A, Analysis of TH expression by immunohistochemistry (top panels) and in situ hybridization (bottom panels) in striatum (St) and SN of saline-treated (MPTP untreated; left) or MPTP-treated (center and right) animals. B, Densitometric measurements of TH immunoreactivity in striata of MPTP-treated mice. In each experiment, all of the data from MPTP-treated animals were normalized to saline-treated wild-type (Wt) animal levels; n = 9 for saline-treated mice, n = 29 for wild-type MPTP-treated group and n = 30 for transgenic (Tg) MPTP-treated group. C, D, Stereological estimations of TH-positive neurons (C) and Nissl-positive cells (D) in the SNpc of untreated and MPTP-treated transgenic and nontransgenic animals 7 d after acute MPTP. The estimated Nissl-stained cell number was as follows: 22915 ± 972 in untreated wild-type; 17388 ± 698 in MPTP-treated wild-type; and 20960 ± 815 in MPTP-treated transgenic animals). The asterisk indicates statistically significantly different with respect to the other two groups. C, D, The number of experiments are as follows: 8 saline-injected wild-type, 10 MPTP-treated wild-type, and 11 MPTP-treated transgenic animals. Statistical analysis was done using one-way ANOVA. Error bars represent SEM.

Figure 5.

Dopamine released from striatum in MPTP-treated young adult mice. A, Amperometric signals attributable to oxidation of dopamine released from striatal slices of a wild-type (Wt) animal (left, top) or a transgenic (Tg) littermate (right, top) after exposure to an external solution with 66 mm K⁺. Dopamine released from wild-type (left, bottom) or transgenic (right, bottom) striatal slices after MPTP treatment. B, Quantification of dopamine released from striatum after MPTP treatment. In each group (untreated or MPTP-treated) the data from transgenic animals were normalized to the respective wild-type values. n = 8 pairs for the untreated group, and n = 17 pairs for MPTP-treated group. Statistical analysis was done using Student's t test. ∗p < 0.05. Error bars represent SEM.

Figure 6.

Attenuation of MPTP-induced dopaminergic striatal denervation by G6PD overexpression in aged mice. A, Representative Western blot showing the decrease of TH expression in striatum after MPTP treatment in young or aged wild-type mice. Sal, Saline; Tub, α-tubulin. B, Quantification of decrease in TH levels in wild-type MPTP-treated animals with respect to age-matched untreated mice by Western blot analysis. Young mice, n = 10 for untreated, and n = 15 for MPTP-treated animals. Aged mice, n = 12 for untreated mice, and n = 16 for the group treated with MPTP. Statistical analysis was done using Student's t test. Error bars represent SEM. C, Box diagram comparing distribution of values of dopamine released from striata of young and old transgenic animals after normalization with their respective MPTP-treated wild-type littermates.