Fig. 3. Mitochondrial antioxidants and calcium modulators attenuate
the toxic cascade in DJ-1 mutant dopaminergic neurons. (A and
B) Oxidized DA in homozygous DJ-1 mutant (hom) neurons treated with
(A) mito-TEMPO or (B) NAC compared to vehicle (veh) at d70 (n = 3).
(C) GCase and a-i-2-sulf activity in lysosomal fractions from homozygous
DJ-1 mutant neurons treated with mito-TEMPO or vehicle at d70 (n = 3).
(D) Lysosomal proteolysis in homozygous DJ-1 mutant neurons treated
with NAC or vehicle at d180 (n = 3). a.u., arbitrary units. (E) Oxidized DA in
homozygous DJ-1 mutant, heterozygous DJ-1 carrier (het), and control
neurons treated with isradipine, FK506, or vehicle (DMSO, dimethyl sulfoxide)
at d90 (n = 3 to 6). (F and G) Immunoblot analysis of a-synuclein (syn211
antibody) at d70 in Triton X–100 (T)–soluble neuronal lysates from (F) control,
heterozygous DJ-1 carrier, and two homozygous DJ-1 mutant lines (n = 4) or (G)
a gene-edited DJ-1 KO line (n = 3 or 4). b-III-tubulin, synapsin, and GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) were used as loading controls.
(H) T-insoluble a-synuclein (C20 antibody) at d70 in homozygous DJ-1 mutant
neurons treated with NAC or vehicle. CBB was used as a loading control (n = 4).
(I) Homozygous DJ-1 mutant neurons expressing mito-roGFP treated with AMPT
at d50 (n = 3). (J and K) Homozygous DJ-1 mutant neurons treated with AMPT
and analyzed at d70 for (J) oxidized DA by nIRF (n = 3) or (K) T-soluble a-synuclein
(syn211 antibody). GAPDH and b-III-tubulin were used as loading controls (n = 3).
Treatment was applied for 30 days (A) to (C), (E), and (H) to (K) or 140 days
(D). Error bars, means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test
(A) to (D) and (G) to (K) or one-way ANOVA with Tukey post hoc test (E) and (F).