Predicting structural features of selected flavonoids responsible for neuroprotection in a Drosophila model of Parkinson's disease

Abstract

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief, thereby underscoring the need for novel strategies to combat disease onset and progression. We have employed an environmental toxin-induced Drosophila Parkinson's disease (PD) model as an inexpensive in vivo screening platform to explore the neuroprotective potential of selected dietary flavonoids. We have identified a specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes involving reduced survival, mobility defects, and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on our screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties through nuclear factor erythroid 2-related factor 2 (Nrf2) but also requires regulation of the immune deficiency (IMD) pathway involving NFκB and the negative regulator poor Imd response upon knock-in (Pirk). Our data have identified specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions.

Keywords: Drosophila; Flavonoid; Neuroinflammation; Neuroprotection; Parkinson’s disease.

Figure 1.. Effects of different groups of flavonoids against paraquat-induced toxicity in a Drosophila PD model.

(A) Schematic representation of the pre- and co-feeding regimen. In the pre-treatment mode, adult male flies were either pre-fed for 4 days with 2.5% sucrose or specified flavonoids followed by continuous exposure to 5 mM PQ and were scored daily for survival. In the co-treatment mode, adult male flies were exposed to both flavonoids and 5 mM PQ at the same time and were scored daily for survival. Survival was scored upon PQ exposure in both pre- and co-treatment regimes and Day 0 in the figure is when PQ exposure starts (red arrow). Survival data for the pre- and co-feeding experiments are representative of ten independent biological replicates with 10 male flies per feeding conditions. (B) Survival assays were performed using adult male flies pre-fed with sucrose or specified flavones followed by continuous exposure to 5 mM PQ as outlined above and the number of live flies was recorded every 24 h until all of the flies were dead and the average survival percentages were plotted. Statistical significances between the PQ-fed and the flavone pre-fed groups followed by PQ exposure were determined using the log-rank test (Suc>PQ vs Nobiletin>PQ, Suc>PQ vs Sinensetin>PQ ***p<0.001; Suc>PQ vs Apigenin>PQ **p<0.01). (C) Survival assays were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavones and the number of live flies was recorded every 24 h and the average survival percentages were plotted. Statistical significances between the PQ-fed and the co-fed PQ+flavone groups were determined using the log-rank test ***p<0.001. (D) Survival assays were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavonols and the number of live flies was recorded every 24 h and the average survival percentages were plotted. No statistical significances between the PQ-fed and the flavonol-fed groups were observed using the log-rank test. (E) Survival assays were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavonones and the average survival percentages were plotted. No statistical significances between the PQ-fed and the flavonone-fed groups were observed using the log-rank test.

Figure 2.. Feeding assays to quantitate food uptake upon exposure to different flavonoids.

Adult male flies were fed either sucrose or specified flavonoids (A) flavones (B) flavonones or flavonols mixed with the blue food dye (1% FD&C Blue#1) and the dye content was measured spectrophotometrically at 630 nm. Data are representative of three independent biological replicates using10 male flies per group. Error bars indicate the standard deviation. No statistical significances between the control sucrose-fed group and the specified flavonoid-fed groups were observed using the Student’s t-test. p>0.05.

Figure 3.. Effects of different groups of flavonoids against paraquat-induced mobility defects.

Negative geotaxis assays were used to determine the effect of flavonoids on the climbing abilities of flies exposed to PQ. The number of flies able to cross 5 cm within 20 s were recorded and plotted at 48 h post PQ exposure. Data are representative of at least five independent experiments involving three to five technical repeats using10 male flies per group. (A) Adult male flies were either pre-fed for 4 days with 2.5% sucrose or flavones (nobiletin, sinensetin and apigenin) followed by continuous exposure to 5 mM PQ and the % climbing index was plotted at 48 h. (B) Mobility assays were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavones (eupatilin, luteolin, and tangeretin) and data were analyzed at 48 h. The protective effects of specified flavonoids on the climbing abilities of flies exposed to PQ were determined by mobility assays. (C) flavonols (fisetin, kaempferol, myricetin, quercetin) and D. flavonones (hesperitin, hesperidin, naringenin, naringin). **p < 0.01; ***p < 0.001 based on one-way ANOVA between indicated feeding conditions.

Figure 4.. The protective effect of flavones against PQ toxicity is not solely dependent on antioxidant activities.

Effects of flavones on the transcript levels of cncC, the human Nrf2 orthologue, and its downstream target gstD1 in Drosophila using qRT-PCR. (A) Adult male flies were either pre-fed for 4 days with 2.5% sucrose or flavones (nobiletin, sinensetin and apigenin) followed by RNA isolation from the heads of adult flies and processed for qRT-PCR. The transcript levels of cncC and gstD1 were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc) flies (assigned a value of 1). (B) Adult male flies were either pre-fed for 4 days with 2.5% sucrose or flavones (nobiletin, sinensetin and apigenin) followed by PQ exposure for 24 h. Total RNA was isolated from the heads of adult flies and processed for qRT-PCR. The transcript levels of cncC and gstD1 were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc>Suc) flies (assigned a value of 1). **p < 0.01; ns: not significant between different feeding conditions based on Mann-Whitney U test. (C) Relative gene expression using qRT-PCR were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavones (eupatilin, luteolin and tangeretin) for 24 h. The transcript levels of cncC and gstD1 were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc) flies (assigned a value of 1). Data represents three independent biological replicates including three technical repeats per specified feeding condition and compared to the control sucrose-fed flies (assigned a value of 1) for data analysis. *p<0.05; **p < 0.01 between different feeding conditions based on the Mann-Whitney U test.

Figure 5.. A specific group of flavones confer protection against PQ-induced neurotoxicity through modulation of the neuroinflammatory responses in Drosophila.

(A) Effects of different flavones on the transcript levels of relish, the human NFκB orthologue in Drosophila using qRT-PCR. A. Adult male flies were either pre-fed for 4 days with 2.5% sucrose or flavones (nobiletin, sinensetin and apigenin) followed by PQ exposure for 24 h. Total RNA was isolated from the heads of adult flies and processed for qRT-PCR. The transcript levels of relish were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc>Suc) flies (assigned a value of 1). **p < 0.01; ***p < 0.001 between different feeding conditions based on Mann-Whitney U test. (B) Relative gene expression using qRT-PCR were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavones (eupatilin, luteolin and tangeretin). The transcript levels of relish were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc) flies (assigned a value of 1). Data represents three independent biological replicates including three technical repeats per specified feeding condition and compared to the control sucrose-fed flies (assigned a value of 1). Statistical differences between the specified feeding groups were analyzed using the nonparametric Mann-Whitney U test **p < 0.01; ***p < 0.001. (C) Effects of different flavones on the transcript levels of pirk, the negative regulator of the IMD pathway in Drosophila using qRT-PCR. A. Adult male flies were either pre-fed for 4 days with 2.5% sucrose or flavones (nobiletin, sinensetin and apigenin) followed by RNA isolation from the heads of adult flies and processed for qRT-PCR. The transcript levels of pirk were plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc>Suc) flies (assigned a value of 1). **p < 0.01; ***p < 0.001 between different feeding conditions based on Mann-Whitney U test. (D) Relative gene expression using qRT-PCR were performed using the co-feeding mode in which adult male flies were exposed to both 5 mM PQ or specified flavones (eupatilin, luteolin and tangeretin). The transcript levels of pirk were analyzed and plotted after normalization with rp49 levels as the internal control. Each data point represents mean ± SEM. The mRNA fold changes are normalized to the sucrose-fed (Suc) flies (assigned a value of 1). Data represents three independent biological replicates including three technical repeats per specified feeding condition and compared to the control sucrose-fed flies (assigned a value of 1). Statistical differences between the specified feeding groups were analyzed using the nonparametric Mann-Whitney U test **p < 0.01; ***p < 0.001.

Figure 6.. Schematic model of flavone-mediated protection against PQ in a Drosophila PD model.

Exposure to PQ induces PD symptoms involving increased mortality, mobility defects and progressive demise of dopaminergic neurons by activating oxidative stress and neuroinflammatory responses, including NFκB in adult male flies. A specific group of flavonoids known as flavones confer protection against PQ-induced neurotoxicity in pre- and co-feeding modes. Our data suggest that certain structural features of flavones including the presence of α,β-unsaturated carbonyl at the C2-C3 position of the flavonoid skeleton and the absence of a functional group substitution at the C3 position are essential to confer protection against PQ-mediated toxicity in Drosophila.

Figure 7.. The comparison of structures of soft electrophiles with antioxidant and anti-inflammatory effects.

(A) Nobiletin (flavone). (B) 13-keto-9Z,11E-octadecadienoic acid (13-oxoHODE; 13-EFOX-L2; fatty acid derivative). (C) Dimethyl fumarate. Unsaturated α,β-carbonyl group is marked with red.