Cellular response to MPTP in C57BL/6 and SW SN. (a) Immunoblot of GSTπ in C57BL/6 (C) and SW (S) SN after saline (control), four doses of 20 mg/kg MPTP, and four doses of 20 mg/kg MPTP plus 12 h. GAPDH was used as load control. Relative quantitation is shown in Right (*, P ≤ 0.01; #, P ≤ 0.05). (b) Immunofluorescent labeling of GSTπ (green) in the SN of C57BL/6 (C57) and SW (same conditions as in a) mice. Each Inset shows a magnified image contained within the field of view. (c) Immunofluorescent labeling of basal levels of phospho-c-Jun (p-cJUN, green) in C57BL/6 and SW (SN neurons are identified by TH, red) after administration of 80 mg/kg MPTP. [Scale bars: b (applies to b and c), 70 μm; b Inset (applies to all Insets), 25 μm.]

MPTP induced neuronal loss in the SN of GSTπ (+/+), GSTπ (+/−), and GSTπ (−/−) mice. (a–c) Representative midbrain sections showing SN immunostained for TH and counterstained for Nissl in GSTπ (+/+), GSTπ (+/−), and GSTπ (−/−) mice 7 days after MPTP. (d) Stereological analysis of the number of dopaminergic neurons within the SN of GSTπ (+/+), GSTπ (+/−), and GSTπ (−/−) mice 7 days after MPTP. (e–g) HPLC measurement of dopamine (e), 3,4-dihydroxyphenylacetic acid (f), and homovanillic acid (g) in striatum from GSTπ (−/−), control, and MPTP-treated mice. (h) HPLC measurement of MPP⁺ from SN of SW and GSTπ (−/−) mice 15 min after four individual i.p. injections (spaced at 2-h intervals) of MPTP (F = 16.73, P ≤ 0.001; *, P ≤ 0.05, SW compared with GSTπ (−/−) SN). (i) Measurement of hydroxyl radical formation in the SN of SW and GSTπ (−/−) mice at baseline and 15 min after three injections of MPTP (F = 25.8, P ≤ 0.0001).

Expression of GST in SW and C57BL/6 brain. (a) RT-PCR of GSTα, GSTμ, and GSTπ mRNA in cerebral cortex (Ctx), striatum (Str), and SN. (b Upper) In situ hybridization of GSTμ and GSTπ in C57BL/6 brain in region of SN (arrows). (b Lower) Enlargement of boxed region showing SN (arrows). (c) Immunoblot of GSTα, GSTμ, and GSTπ in SN, striatum, cerebral cortex, and liver between mouse strains. (d and e) GSTμ and GSTπ expression in the SN. TH identifies dopaminergic neurons. (f) GSTπ and TH in normal-aged human SN. [ Scale bar: f (applies to d–f), 25 μm.]

GST inhibition increases dopaminergic neuron death in an MPTP-resistant strain. (a) SN neuron, in vitro, immunostained for TH (red) and GSTπ (green). The coexpression of the two fluorophores is seen as yellow. (b) EA (50 nM) treatment before MPP⁺ (50 nM for 2 days) resulted in 30 ± 4% TH-positive cell loss compared with 0% with MPP⁺ or 16 ± 12% with EA alone (F = 5.36, P ≤ 0.05). (c) GSTπ inhibition (50 nM) with MPP⁺ (50 nM for 2 days) resulted in 49 ± 9% dopamine cell loss, compared with 0% loss in nontransfected cultures treated with MPP⁺ (F = 5.23, P ≤ 0.005) and 26 ± 11% loss with GSTπ siRNA inhibition alone (not significant). (Scale bar: 25 μm.)