park+/+ and park-/- Drosophila have sexually dimorphic brain redox chemistry

Abstract

Sexual dimorphism in Parkinson's disease (PD) pathophysiology is poorly understood. Elucidating consequences of disease-causing mutations on brain redox chemistry may reveal therapeutic targets for all people with PD. We report that male Drosophila had increased hydrogen peroxide and glutathione (G-SH) redox disequilibrium in vulnerable dopaminergic neuron mitochondria. Levels of cysteine and oxidized cystine were decreased, with cysteine/cystine ratios (indicating less oxidative stress) and G-SH levels being elevated in parkin-null (park-/-) Drosophila brains, and more so in males. We report effects of parkin loss and sex on the levels of low-molecular-weight thiols involved in G-SH synthesis, providing clues as to mechanisms implicated in altered levels of brain G-SH, cysteine and cystine. Protein nitration was decreased in the brain of park-/- flies, especially in females, suggesting that decreased nitric oxide levels compensate for loss of parkin or lack of protective nitric oxide synthase activity. Our results imply that park-/- flies have elevated levels of G-SH that meet antioxidant demand in the absence of parkin in the whole brain, but not in vulnerable neurons. Identification of sexually dimorphic PD risk factors could inform symptom management and highlight sex-specific therapeutic strategies.

Keywords: Drosophila; Antioxidants; Dopaminergic neuron; Glutathione; Hydrogen peroxide; Mitochondria; Oxidative stress; Parkin; Parkinson's disease; Redox; Sex; Sexual dimorphism.

Fig. 1.

Decreases in climbing capability and motivation in park^−/− Drosophila are accompanied by elevated levels of PPL1 mitochondrial hydrogen peroxide. (A) Average height climbed and climbing attempts were recorded for individual flies over 20 min. Each data point represents data from one park^+/+ or park^−/− fly (n for height climbed: park^+/+ females=38; park^−/− females=35; park^+/+ male=32; park^−/− male=41; n for climbing attempts: park^+/+ females=37; park^−/− females=32; park^+/+ male=33; park^−/− male=38). Data were collected from 20 cohorts. (B) Contingency graphs demonstrate that fewer female park^−/− flies attempt to climb (n=30 for park^+/+ females, n=24 for park^+/+ males, n=34 for park^−/− females, n=29 for park^−/− males). Data were collected from eleven cohorts. (C) Representative images of PPL1 regions from female (F) or male (M) flies expressing mito-roGFP2-Orp1. Brains were dissected on days 4-6 post eclosion, and ratios of total volumes of oxidized to non-oxidized fluorophore emissions were calculated for one PPL1 region per brain. Top row: Raw images showing PPL1 regions stained for tyrosine hydroxylase (red) in flies expressing TH-driven mito-roGFP2-Orp1. Middle and bottom rows: Selected volume isosurfaces of oxidized (blue, middle row) and non-oxidized (green, bottom row) mito-roGFP2-Orp1 above threshold. Scale bar: 10 µm. (D) Each data point represents the ratio of the total oxidized/non-oxidized roGFP2 volume from one PPL1 region (n=25 for park^+/+ females, n=28 for park^−/− females, n=31 for park^+/+ males, n=23 for park^−/− males). Data represent five different dissection/staining experiments. Two-way ANOVA followed by Tukey's multiple comparisons tests were used for A and D. Fisher's exact tests were performed to determine effect of genotype and sex (B). Error bars represent standard error of the mean; Fisher's exact test and post-hoc P values are indicated above graphs. ANOVA P values are indicated to the right of graphs in A and below graphs in D. In panels A, B and D, circles indicate female flies; squares indicate male flies; white circles/squares indicate park^−/− flies; black circles/squares indicate park^+/+ flies.

Fig. 2.

Vulnerable PPL1 neurons in park^+/+ and park^−/− males, and park^−/− females have glutathione disequilibrium, while whole-brain oxidation indicators are decreased. (A) Representative images of PPL1 regions from flies expressing mito-roGFP2-Grx1. Brains were dissected on days 4-6 post eclosion and ratios of total volumes of oxidized to non-oxidized fluorophore emissions were calculated for one PPL1 region per brain. Top row: raw images showing PPL1 regions stained for tyrosine hydroxylase (red) in flies expressing TH-driven mito-roGFP2-Grx1. Middle and bottom rows: selected volume isosurfaces of oxidized (blue, middle row) and non-oxidized (green, bottom row) mito-roGFP2-Grx1 above threshold. Scale bar: 10 µm. (B) Each data point represents the ratio of the total oxidized/non-oxidized total roGFP2 volume from one PPL1 region (n=18 for control (park^+/+) females, n=23 for parkin-null (park^−/−) females, n=22 for park^+/+ males, n=17 for park^−/− males). Data represent eleven different dissection/staining experiments. (C) park^+/+ and park^−/− males, and park^−/− females have elevated antioxidant markers at the whole-brain level. park^+/+ and park^−/− fly heads were collected and frozen on days 4-6 post eclosion. LC-MS/MS was performed to detect reduced levels of glutathione (G-SH), cysteine and cystine. The increased ratio of cysteine to cystine in park^−/− males and females indicates a more-reduced redox environment. Each data point represents one tube of homogenate from a pool of about 20 brains (n=11 for park^+/+ females, n=8 for park^−/− females, n=11 for park^+/+ males, n=8 for park^−/− males). Samples for each genotype were continuously harvested for 1 year. LC-MS/MS was performed on three groups of samples on three different dates. (B,C) Two-way ANOVA followed by Tukey's multiple comparisons test were performed to determine effect of genotype and sex. Error bars represent standard error of the mean. ANOVA and post-hoc P values are indicated below and above graphs, respectively. In panels B and C, circles indicate female flies; squares indicate male flies; white circles/squares indicate park^−/− flies; black circles/squares indicate park^+/+ flies.

Fig. 3.

Transsulfuration pathway components are disrupted in brains of park^+/+ and park^-/- male, and park^−/− females. (A) Transsulfuration pathway of glutathione (G-SH) synthesis. Measured low-molecular-weight (LMW) thiols are within blue ovals. Blue arrows indicate direction of change in female and male park^−/− flies (P) and park^+/+ males (♂). Enzymes are italicized. Essential amino acid methionine and tetrahydro-folate from dietary folate feed into the transsulfuration pathway of G-SH synthesis. G-SH is transported out of the cell where it is broken down into cysteinylglycine (cysteine-gly) dimers, which are further broken down to cysteine and glycine (gly) by dipeptidase. Amino acids are transported into the cell where they can enter the transsulfuration pathway as indicated by the arrows. Solid arrows indicate enzymatic reactions; dashed arrows indicate movement across the plasma membrane; gly, glycine; glu, glutamate. (B) park^+/+ and park^−/− males, and park^−/− females have elevated cystathionine, and female and male park^−/− flies have decreased cysteine, cystine (CySS, oxidized cysteine), and γ glutamylcysteine. park^+/+ and park^−/− fly heads were harvested and frozen on days 4-6 post eclosion. Each data point represents one tube of homogenate from a pool of approximately twenty brains (n=11 for park^+/+ females, n=8 for park^−/− females, n=11 for park^+/+ males, n=8 for park^−/− males). Samples for each genotype were continuously harvested for one year; LC-MS/MS was performed on three groups of samples on three different dates. Two-way ANOVA followed by Tukey's multiple comparison's tests were performed to determine effect of genotype and sex. Error bars represent standard error of the mean; ANOVA and post-hoc P values are indicated below and above graphs, respectively. In panel B, circles indicate female flies; squares indicate male flies; white circles/squares indicate park^−/− flies; black circles/squares indicate park^+/+ flies. Created in BioRender. Buhlman, L. (2025) https://BioRender.com/qti62ct.

Fig. 4.

Central brain nitration levels are elevated in park^+/+ and park^−/− males, and decreased in park^−/− females. Brains were dissected and total volume of central brain anti-tyrosine nitration antibody emission was calculated for flies aged 4 to 6 post eclosion. (A) Representative 2D-images of the central brain (encircled) obtained from female (F) or male (M) park^+/+ and park^−/− flies. Blue isosurfaces indicate anti-nitrotyrosine antibody staining above background. Top row: raw images. Middle row: whole-brain antibody emission above background. Bottom row: central brain antibody emission above background. Scale bar: 200 μm. (B) Top graph: Central brain volume is decreased in park^+/+ and park^−/− males, and park^−/− females. Bottom graph: Brain nitration is decreased by the park mutation and by the female sex. Each point represents data for one brain (for brain size data, n=11 for park^+/+ females, n=15 for park^−/− females, n=18 for park^+/+ males, n=12 for park^−/− males; for nitration data, n=10 for park^+/+ females, n=9 for park^−/− females, n=18 for park^+/+ males, n=11 for park^−/− males). Data represent five different dissection/staining experiments. Circles indicate female flies; squares indicate male flies; white circles/squares indicate park^−/− flies; black circles/squares indicate park^+/+ flies. Two-way ANOVA followed by Tukey's multiple comparisons tests were performed to determine the effect of genotype and sex. Error bars represent standard error of the mean; ANOVA and post-hoc P values are indicated to the right and above graphs, respectively.

Fig. 5.

DNOS levels are unaffected by sex or parkin loss of function. (A) Representative 2D-images of the central brain (encircled) showing the dNOS-driven EGFP volume. Brains were dissected from day 4 to 6 post-eclosion park^+/+ and park^−/− flies expressing dNOS-driven EGFP, and the total volume of central brain EGFP emission was calculated. Green ‘isosurfaces’ indicate EGFP expression above background. Top row: raw images from flies expressing NOS-driven GFP. Middle row: EGFP expression above background. Bottom row: Total volume of central brain EGFP expression. Scale bar: 200 μm. (B) Plotted is the total fluorescence volume per central brain. Each point represents data from one fly (n=11 for park^+/+ females, n=15 for park^−/− females, n=18 for park^+/+ males, n=12 for park^−/− males). Circles indicate female flies; squares indicate male flies; white circles/squares indicate park^−/− flies; black circles/squares indicate park^+/+ flies. Data represent five different dissection/staining experiments. Two-way ANOVA following by Tukey's multiple comparisons tests were performed to determine the effect of genotype and sex. Error bars represent standard error of the mean; ANOVA and post-hoc P values are indicated below and above graphs, respectively.