Neuroprotective Potential of Eugenol in Polyglutamine-Mediated Neurodegenerative Disease Using Transgenic Drosophila Model

Abstract

Polyglutamine (PolyQ) diseases including Huntington's disease are devastating neurodegenerative disorders characterized by progressive neuronal loss and motor dysfunction. PolyQ pathology involves multiple cellular events and phytochemicals with multi-target mechanisms hold promise to treat these diseases with least side effects. One such promising phytochemical is Eugenol, which possesses antioxidant and anti-inflammatory properties, potentially targeting disrupted cellular pathways in PolyQ diseases. The present study investigated the effects of Eugenol on neurodegeneration and motor dysfunction in transgenic Drosophila models of PolyQ diseases. In this study, the robust pseudopupil assay was performed to analyze adult photoreceptor neuron degeneration, a marker of widespread degenerative events. Furthermore, the well-established crawling and climbing assays were conducted to evaluate progressive motor dysfunction in the PolyQ larvae and flies. This study found that Eugenol administration at disease onset or after progression reduced PolyQ disease phenotypes, particularly, neurodegeneration and motor dysfunction in a dose-dependent manner and with no side effects. Thus, this study suggests that Eugenol could be a viable candidate for developing treatments for PolyQ diseases, offering a multi-target approach with the potential for minimal or no side effects compared to conventional therapies.

Keywords: Drosophila; Eugenol; Huntington’s disease; neurodegenerative disease; polyglutamine (PolyQ) disease.

Graphical Abstract

Figure 1.

Eugenol doses have no effect on food intake. Various doses of Eugenol were fed to third instar larvae. No differential feeding behavior was observed in non-pathogenic (A) Httex1p Q20 as well as pathogenic (B) Httex1p Q93 larvae. Two groups of 10 larvae were assayed (n = 20) for 6 spectrophotometer readings. The box plots denote the median, minimum and maximum values. Data was analyzed using an analysis of variance (ANOVA).

Figure 2.

Administration of Eugenol at the disease onset suppresses photoreceptor degeneration in PolyQ flies. No neurotoxicity was observed in 7-day-old-non-pathogenic Httex1p Q20 flies (A). An effective dose of 20 μM Eugenol significantly suppressed neurodegeneration, when fed since early larval stage in pathogenic Httex1p Q93 (B, D, F) and Q48 flies (C, E, G) at 7-day post eclosion. We quantified these data as the number of rhabdomeres per ommatidium (D, E) and the distribution of the percent of ommatidia containing the specified number of photoreceptors (F, G). For each condition, 6 flies were assayed and at least 300 ommatidia were scored. The box plots show the median, minimum and maximum values. Data analysis was performed using an analysis of variance (ANOVA) followed by Tukey’s post hoc test, ***, P < 0.001; **, P < 0.01, compared with control.

Figure 3.

Suppression of photoreceptor neurodegeneration in PolyQ flies by Eugenol after disease progression. Feeding Eugenol at an effective dose of 20 μM only during adult stage suppressed photoreceptor neurodegeneration in Httex1p Q93 (A, C, E) and Q48 (B, D, F) flies. Data was analyzed by calculating the number of rhabdomeres per ommatidium (C, D) and the distribution of the percent of ommatidia containing the specified number of photoreceptors (E, F). A total of 6 flies were assayed per condition and at least 300 ommatidia were scored. The box plots show the median, minimum and maximum values. Data was analyzed by an analysis of variance (ANOVA) followed by Tukey’s post hoc test, ***, P < 0.001; **, P < 0.01; *, P < 0.05, compared with control.

Figure 4.

Administration of Eugenol improves locomotor dysfunction of PolyQ larvae. Feeding 20 μM Eugenol significantly suppressed impaired motor function of Httex1p Q93 (A) and Q48 (B) larvae. For each condition, the crawling ability of 10 larvae (n = 10) was monitored and the experiment was repeated 2 times for each larva. The box plots show the median, minimum and maximum values. Data analysis was done using an analysis of variance (ANOVA) followed by Tukey’s post hoc test, ***, P < 0.001; **, P < 0.01, as compared to control.

Figure 5.

Eugenol ameliorates locomotor dysfunction of PolyQ flies. (A) Eugenol had no effect in non-pathogenic Httex1p Q20 condition. Feeding Eugenol since larval period (B) and (C), or only during adult life (D) and (E), markedly suppressed locomotor dysfunction of diseased Httex1p Q93 and Q48 flies at 7-day post eclosion. The climbing ability of 2 groups of 10 flies (n = 20) were assayed per condition for a total of 6 trials. Data was analyzed using Kruskal-Wallis test followed by Mann-Whitney test with Bonferroni correction to adjust P-values; values represent mean ± SEM (**, P < 0.01; *, P < 0.05, compared with control).