Mucuna pruriens (Velvet bean) rescues motor, olfactory, mitochondrial and synaptic impairment in PINK1B9 Drosophila melanogaster genetic model of Parkinson's disease

Abstract

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3-6 (I), 10-15 (II) and 20-25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effects of Mpe and L-Dopa on lifespan.

(A): Lifespan, expressed as % survival rates, of wild type (WT) and PINK1B9 flies. (B) and (C): Lifespan of PINK1B9 treated with Mucuna pruriens extract (Mpe) 0.1%,only when adults (L−/A+) (panel B) or from their larval stage to the end of their life-cycle (L+/A+) (panel C), respectively, as compared to lifespan of untreated PINK1B9 flies. (D): Lifespan of PINK1B9 flies treated with L-Dopa (L+/A+) 0.01%. *indicates p<0.05 at Kaplan-Meier survival curves (Gehan-Breslow–Wilcoxon - GraphPad Prism 5.01) between WT and untreated PINK1B9 (A) and between untreated PINK1B9 and PINK1B9 fed Mpe 0.1% (C).

Figure 2. Effects of Mpe and L-Dopa on climbing activity.

(A): Climbing activity of adult males wild-type (WT), untreated PINK1B9, PINK1B9 treated with Mucuna pruriens extract (Mpe) 0.1% and PINK1B9 treated with L-Dopa 0.01% (L-Dopa 0.01%). (B): Climbing activity of PINK1B9 adult males treated with Mpe 1 and 10% as compared with WT and untreated PINK1B9. (A) and (B): Treatments were administered to flies from their larval stage to the end of their life-cycle (L+/A+) and their effects were assayed at three different age steps (I: 3–6; II: 10–15; III: 20–25 days) of flies’ life-span. Values are average + SEM. *indicates p<0.05 at two-way ANOVA followed by HSD post-hoc test as compared to WT; **indicates p<0.05 at two-way ANOVA followed by HSD post-hoc test as compared to PINK1B9; ***indicates p<0.05 at two-way ANOVA followed by HSD post-hoc test as compared to PINK1B9 Mpe 0.1%. (C) and (D): Percentages of adult males WT, PINK1B9, Mpe 0.1%, L-Dopa 0.01% (C) and Mpe 1 and 10% (D) that could climb unto, or above, the line drawn at 6 cm from the bottom of the tube within 10 seconds.

Figure 3. Electroantennogram responses to 1-hexanol.

Histograms in (A) show the dose-response relationship and their differences in signal for olfactory stimulations in WT, untreated PINK1B9 and in Mpe (0.1%)- and L-Dopa (0.01%)-treated PINK1B9, recorded in flies from group II. As odor stimuli, the 1-hexanol was administered in a 3-step dose from 0.01 to 1% in hexane. Values are average + SEM. *indicates p<0.05 at one-way ANOVA followed by HSD post hoc test as compared to the previous concentration of the stimulus. **indicates p<0.05 at one-way ANOVA followed by HSD post-hoc test as compared to WT. (B) Samples of EAGs recordings in response to 1-hexanol 0.1%.

Figure 4. Effects of Mpe and L-Dopa on olfactory behavior.

Responses to 1-hexanol 0.1% and water (H2O) of WT, untreated PINK1B9 and in Mpe (0.1%)- and L-Dopa (0.01%)-treated PINK1B9 flies. Values are average + SEM. *indicates p<0.05 at two-way ANOVA followed by HSD post hoc test as compared to WT, PINK1B9 Mpe 0.1%, PINK1B9 L-Dopa 0.01%.

Figure 5. Effects of Mpe on T-bars and mitochondria in antennal lobes and thoracic ganglia.

Transmission electron microscopy (TEM) images of T-bars and mitochondria inside antennal lobes (ALs) of wild type (WT), untreated PINK1B9 and in Mpe (0.1%)-treated PINK1B9 flies. (A): T-bars in a presynaptic bouton of PINK1B9 ALs (arrowheads). Asterisks indicate mitochondria inside presynaptic boutons and neurites. Inset: high magnification of two T-bar in coronal section. (B): T-bars in presynaptic boutons of ALs of PINK1B9 Mpe 0.1% (arrowheads). Asterisks indicate mitochondria inside presynaptic boutons and neurites. (C): swelling on the external mitochondrial membrane (at high magnification in the inset) and mitochondrial cristae widely degenerated (asterisks) in ALs of PINK1B9. (D): Mitochondria of PINK1B9 Mpe 0.1% (asterisks). (E): Presynaptic T-bar density in ALs of WT, PINK1B9 and PINK1B9 0.1% Mpe flies. Values are average + SEM. *indicates p<0.01 at two tailed t-test with respect to PINK1B9. (F): T-bar density in thoracic ganglia of WT, PINK1B9 and PINK1B9 0.1% Mpe flies. Values are average + SEM. *indicates p<0.01 at two tailed t-test with respect to PINK1B9. (G): Percentages of damaged mitochondria in ALs of WT, PINK1B9 and PINK1B9 0.1% Mpe flies. Values are average + SEM. *indicates p<0.01 at two tailed t-test with respect to PINK1B9. Abbreviations: postsyn: postsynaptic; presyn: presynaptic. Scale bars are 200 µm in A and B and 500 µm in C and D.

Figure 6. Effects of Mpe and L-Dopa on BRP and TH.

(A): Representative western blot analysis of head homogenates from adult wild type (WT), untreated PINK1B9 and in Mpe (0.1%)- and L-Dopa (0.01%)-treated PINK1B9 flies showing labeled bands of Bruchpilot protein (BRP), of Tyrosine hydroxylase (TH) and of the loading control actin (from top to bottom). (B): Percentage of protein expression variation of BRP and TH in WT, untreated PINK1B9 and in Mpe (0.1%)- and L-Dopa (0.01%)-treated PINK1B9 flies. Values are average + SEM. *indicates p<0.05 at one-way ANOVA with respect to WT; **indicates p<0.05 at one-way ANOVA (HSD post-hoc test) with respect to PINK1B9 Mpe 0.1%.