Fig 2: Validation of the p47phox-NOX2 proximity ligation assay as an index of NOX2 activity in situ.(A) p47phox-NOX2 PL signal (red) and DHE-related fluorescent signal (green) in pVMB cultures. Cells treated for 24 h with a sublethal concentration (50 nM) of the complex I inhibitor, rotenone, showed increased PL signal for p47phox-NOX2 interaction and increased cytosolic superoxide production detected as DHE fluorescence. This cellular response to rotenone exposure was prevented in co-treatment with Nox2ds-tat, but not by the scrambled variant, Scr. Nox2ds-tat. (B, C) Quantification of the p47phox-NOX2 PL signal and DHE-related fluorescence intensity in pVMB cultures. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100) analyzed for each independent experiment (100–150 neurons/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni’s correction (n = 3 independent experiments). (D) Similarly, a significant increase of PL signal for p47phox-NOX2 interaction and DHE fluorescence was observed in wild-type HEK-293 cells exposed to rotenone for 24 h. Both signals for p47phox-NOX2 interaction and DHE were prevented in CRISPR/Cas9 gene-edited NOX2−/− HEK-293 cells and by co-treatment with Nox2ds-tat in wild-type HEK-293. Scr. Nox2ds-tat co-treatment failed to prevent the rotenone induced increase in p47phox-NOX2 interaction and DHE fluorescence. (E, F) Quantification of the p47phox-NOX2 PL signal and DHE-related fluorescence intensity in HEK-293 cells. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100) analyzed for each independent experiment (100–150 cells/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni’s correction (n = 4 independent experiments) In plots B, C, E and F, ***denotes p < 0.0001 significance compared vehicle; ###denotes p < 0.0001 significance compared to rotenone. (G, H) Dose response of PL p47phox-NOX2 and DHE signals to NOX2 inhibitors. HEK-293 cells were treated with rotenone (50 nM) for 24 h to activate NOX2 in the absence or presence of increasing concentrations of inhibitors (30 nM to 10 μM for both compounds). Quantification of the PL and DHE signals shows that, in a dose-dependent manner, the highly specific NOX2 inhibitors, Nox2ds-tat and CPP11G reduced the p47phox-NOX2 PL signal (black symbols) (IC50: 0.7 μM for Nox2ds-tat and 0.1 μM for CPP11G) paralleled by attenuation of DHE fluorescence (open symbols) (IC50: 1.3 μM for Nox2ds-tat and 0.2 μM for CPP11G; n = 3 independent experiments).

Fig 3: Rotenone induced mitochondrial superoxide elicits NOX2 activation.(A) Representative images of p47phox-NOX2 PL signal (red) in pVMB neurons exposed 4 or 8 h to rotenone (left column), or rotenone + MitoTEMPO (right column). (B) Quantification and time course of PL signal for NOX2 activity induced by rotenone (blue line) or rotenone + MitoTEMPO (red line) exposure. As shown, rotenone elicited NOX2 activation (detected as p47phox-NOX2 PL signal) within 2 h and activation persisted for at least 24 h of rotenone exposure. Co-treatment with the mitochondrial superoxide scavenger, MitoTEMPO (25 nM) prevented NOX2 activity at all time points, indicating that mitochondrial ROS are responsible for NOX2 activation. Symbols represent the normalized average of cellular fluorescence intensities (with vehicle set to 100) analyzed in 3 independent experiments: (100–150 neurons/treatment group). Statistical comparison by one-way ANOVA with Bonferroni’s correction.*denotes p < 0.05 and ***denotes p < 0.0001 significance compared to vehicle; ###denotes p < 0.0001 significance compared to rotenone.

Fig 4: In vivo nigrostriatal NOX2 activation and oxidative damage in the rotenone model of PD are prevented by a brain penetrant NOX2 inhibitor.(A) Representative images of p47phox-NOX2 PL signal (red) after 5 days of rotenone treatment ± CPP11G. Nigrostriatal DA neurons (TH, blue) showed a sustained NOX2 activity (red) that was prevented in animals that received co-treatment with CPP11G. (B) Quantification of p47phox-NOX2 proximity ligation signal. Each symbol represents the normalized intensity of an individual animal (with vehicle set to 100) from which 4 sections /animal and 30–40 cells /section were measured. Comparison by ANOVA with post hoc Bonferroni’s correction. (C) Fluorescence images of 4-hydroxynonenal (4-HNE) immunohistochemistry (gray) as a marker of lipid peroxidation. Five days of rotenone treatment caused a significant increase of 4- HNE in nigrostriatal neurons. Co-treatment with CPP11G prevented lipid peroxidation. (D) Quantification of 4-HNE fluorescence signal. Each symbol (n = 6) represents an individual animal as above. Comparison by ANOVA with Bonferroni’s correction. ***denotes p < 0.0001 significance compared to vehicle; ###denotes p < 0.0001 significance compared to rotenone.

Fig 5: Dopamine neurons show earlier NOX2 activation than microglia in two rat models of PD.(A) Representative images showing p47phox-NOX2 PL signal (red) in DA neurons (TH; blue) and microglia (Iba-1; green) in substantia nigra sections from rats treated with vehicle (first row), rotenone for 1 day (second row), 5 days (third row) or endpoint rotenone treatment (10–14 days; fourth row). Rats treated for 1 day showed activation of NOX2 in nigrostriatal DA neurons but not microglia. Five days of rotenone treatment elicited an increase in p47phox-NOX2 PL signal in both DA neurons and in microglia. A similar NOX2 activity state was detected in endpoint treated rats. (B) Quantification of microglial and neuronal NOX2 activity by PL p47phox-NOX2. Symbols represent the normalized mean intensity (with vehicle being set to 100) analyzed in single animals (n = 6 animals/treatment group: 4 sections/animal and 30–40 cells./Section were measured.). Statistical comparison was performed by one-way ANOVA with Bonferroni’s correction. ***denotes p < 0.0001 significance compared to vehicle for DA neurons; ###p < 0.0001 significance compared to vehicle for microglia. (C) Shown are p47phox-NOX2 PL signal in DA neurons (TH; blue) and microglia (Iba1; green) in the substantia nigra from rats that received a unilateral nigral injection of AAV2 h-a-syn. Three weeks after AAV2 h-a-syn injection (second row), a significant increase in p47phox-NOX2 PL signal, compared to the contralateral un-injected nigra (first row), was observed in DA neurons but not in microglia. Microglial NOX2 activity started to appear after 6 weeks from AAV2 h-a- syn injection (third row), with a maximal activation observed in the AAV2 h-a-syn injected nigra after 12 weeks. (D) Quantification of p47phox-NOX2 PL signal in nigrostriatal dopamine neurons and microglia from control (uninjected) and AAV2 h-a-syn injected rat brain hemispheres. Symbols represent the normalized mean intensity (with the uninjected hemisphere being set to 100) from each hemisphere in each animal (n = 6 animals/time-point) Statistical comparison was performed by one-way ANOVA with Bonferroni’s correction. ***denotes p < 0.0001 significance compared to control for DA neurons. #denotes p < 0.05 and ###denotes p < 0.0001 significance compared to control for microglia.