Neuroprotective actions of a fatty acid nitroalkene in Parkinson's disease

Abstract

To date there are no therapeutic strategies that limit the progression of Parkinson's disease (PD). The mechanisms underlying PD-related nigrostriatal neurodegeneration remain incompletely understood, with multiple factors modulating the course of PD pathogenesis. This includes Nrf2-dependent gene expression, oxidative stress, α-synuclein pathology, mitochondrial dysfunction, and neuroinflammation. In vitro and sub-acute in vivo rotenone rat models of PD were used to evaluate the neuroprotective potential of a clinically-safe, multi-target metabolic and inflammatory modulator, the electrophilic fatty acid nitroalkene 10-nitro-oleic acid (10-NO₂-OA). In N27-A dopaminergic cells and in the substantia nigra pars compacta of rats, 10-NO₂-OA activated Nrf2-regulated gene expression and inhibited NOX2 and LRRK2 hyperactivation, oxidative stress, microglial activation, α-synuclein modification, and downstream mitochondrial import impairment. These data reveal broad neuroprotective actions of 10-NO₂-OA in a sub-acute model of PD and motivate more chronic studies in rodents and primates.

Fig. 1. 10-NO₂-OA activates Nrf2 regulating heme oxygenase-1 expression in dopaminergic neurons of the SNpc.

a Immunohistochemical assay for Nrf2 (Cohort 2, n = 4) reveals a significant nuclear recruitment of Nrf2 in DA neurons both in rats treated with 10-NO₂-OA (45 mg/Kg) and Rotenone + 10-NO₂-OA. A remarkable increase of Nfr2 expression in nigrostriatal DA neurons was also observed in 10-NO₂-OA treatments, whereas rotenone induced downregulation of Nrf2 (Scale bar: 20 μm). b (upper graphic) Quantification of nuclear Nrf2. Symbols represent the percent of the ratio nuclear/total Nrf2 from a single animal (3 slices /brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle and Rotenone). b (lower graphic) Quantification of Nrf2 fluorescence intensity. Symbols represent the normalized means of the intensity (with Vehicle set at 100%) from a single rat (3 slices /brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle, #p < 0.0001 compared to 10-NO₂-OA and Rotenone + 10-NO₂-OA). c Immunohistochemical stain for HO-1 expression (Cohort 2). Rats treated with 10-NO₂-OA (45 mg/Kg) or co-treated with 10-NO₂-OA and rotenone displayed a significant increase of HO-1 protein expression (red) in dopaminergic nigrostriatal neurons (TH, blue—Scale bar: 35 μm). d Quantification of HO-1 fluorescence intensity. Symbols represent the normalized means of the intensity (with Vehicle set at 100) from a single rat (6 slices/brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle and Rotenone).

Fig. 2. 10-NO₂-OA inhibits rotenone mediated accumulation of 4-HNE-α-syn adduct in the SNpc.

a Confocal analysis of the detection of 4-HNE-α-synuclein adducts (PL 4-HNE α-synuclein) in SNpc DA neurons of rats (Cohort 2). Rotenone treatment caused a significant accumulation of 4-HNE-α-synuclein adducts (red) in nigrostriatal neurons (TH, blue) that was inhibited in rats treated with 10-NO₂-OA (45 mg/Kg—Scale bar: 35 μm). b Quantification of PL 4-HNE-α-synuclein. Symbols represent the normalized means of the intensity (with Vehicle set at 100%) from a single rat (6 slices/brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle, ^#p < 0.0001 compared to Rotenone).

Fig. 3. 10-NO₂-OA inhibits NOX2 activation in a sub-acute rotenone model of PD.

a PL assay for in situ detection of NOX2 activation (PL p47^phox:NOX2) (Cohort 2). Rats treated with 10-NO₂-OA (45 mg/Kg) displayed inhibition of rotenone-induced NOX2 activation (red) in dopaminergic neurons of SNpc (TH, blue—Scale bar: 35 μm). b Quantification PL p47^phox:NOX2 interaction. Symbols represent the normalized means of the intensity (with Vehicle set at 100%) from a single rat (6 slices/brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle, ^#p < 0.0001 compared to Rotenone).

Fig. 4. 10-NO₂-OA inhibits rotenone-induced LRRK2 activation in dopaminergic neurons of SNpc.

a PL assay for in situ detection of LRRK2 kinase activity (PL pS1292-LRRK2) (Cohort 2). Rotenone treatment elevated LRRK2 kinase activity (red), which was inhibited by treatment with 10-NO₂-OA (45 mg/Kg) in DA neurons of SNpc (TH, blue—Scale bar: 35 μm). b Quantification of the PL pS1292-LRRK2 fluorescence signal. Symbols represent the normalized means of the intensity (with Vehicle set at 100%) from a single rat (6 slices/brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle, ^#p < 0.0001 compared to Rotenone).

Fig. 5. 10-NO₂-OA inhibits α-synuclein-mediated mitochondrial import impairment in a sub-acute rotenone model of PD.

a Confocal microscopy analysis for PL signal between α-synuclein–TOM20 interaction in SNpc dopaminergic neurons (TH, blue) (Cohort 2). Rotenone treatment of rats induced a strong PL signal for α-synuclein:TOM20 (red). Co-treatment with 10-NO₂-OA (45 mg/Kg) inhibited the rotenone-induced α-synuclein:TOM20 PL signal (Scale bar: 35 μm). b Quantification of the fluorescence signal. Symbols represent the normalized means of the intensity (with Vehicle set at 100%) from a single animal (6 slices /brain). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle, ^#p < 0.0001 compared to Rotenone).

Fig. 6. 10-NO₂-OA inhibits rotenone-induced microglial activation in SNpc (Cohort 2).

a Rats treated with rotenone showed a significant increase in microglial activation (green) in SNpc analyzed immunohistochemically for the microglial activation marker, CD68 (red). Co-administration of 10-NO₂-OA (45 mg/Kg) inhibited rotenone-induced microglial activation (Scale bar main figure: 35 μm; Scale bar ×200 magnification: 15 μm). b Quantification of the signal relative to CD68 in microglia (Iba1) with symbols that represent the normalized means of the intensity (with Vehicle set at 100%) from a single rat (4 slices/brain). The number of animals per treatment group were the following: Vehicle (n = 7), 10-NO₂-OA (n = 9), Rotenone (n = 8), Rotenone + 10-NO₂-OA (n = 10). Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni correction (*p < 0.0001 compared to Vehicle and 10-NO₂-OA, ^#p < 0.0001 compared to Rotenone).