Adult health and transition stage-specific rotenone-mediated Drosophila model of Parkinson's disease: Impact on late-onset neurodegenerative disease models

Abstract

Parkinson's disease (PD) affects almost 1% of the population worldwide over the age of 50 years. Exposure to environmental toxins like paraquat and rotenone is a risk factor for sporadic PD which constitutes 95% of total cases. Herbicide rotenone has been shown to cause Parkinsonian symptoms in multiple animal models. Drosophila is an excellent model organism for studying neurodegenerative diseases (NDD) including PD. The aging process is characterized by differential expression of genes during different life stages. Hence it is necessary to develop life-stage-matched animal models for late-onset human disease(s) such as PD. Such animal models are critical for understanding the pathophysiology of age-related disease progression and important to understand if a genotropic drug/nutraceutical can be effective during late stages. With this idea, we developed an adult life stage-specific (health and transition phase, during which late-onset NDDs such as PD sets in) rotenone-mediated Drosophila model of idiopathic PD. Drosophila is susceptible to rotenone in dose-time dependent manner. Rotenone-mediated fly model of sporadic PD exhibits mobility defects (independent of mortality), inhibited mitochondrial complex I activity, dopaminergic (DAergic) neuronal dysfunction (no loss of DAergic neuronal number; however, reduction in rate-limiting enzyme tyrosine hydroxylase (TH) synthesis), and alteration in levels of dopamine (DA) and its metabolites; 3,4-Dihydroxyphenylacetic acid (DOPAC) and Homovanilic acid (HVA) in brain-specific fashion. These PD-linked behaviors and brain-specific phenotypes denote the robustness of the present fly model of PD. This novel model will be of great help to decipher life stage-specific genetic targets of small molecule mediated DAergic neuroprotection; understanding of which is critical for formulating therapeutic strategies for PD.

Keywords: Drosophila; Parkinson’s disease; dopamine; health phase; rotenone; transition phase.

FIGURE 1

Dose and time-dependent mortality of Drosophila (OK) exposed to rotenone during different phases of adult life. Mortality pattern among adult male flies of early health span (A), late health span (B), and transition phase (C), exposed to seven different concentrations of rotenone (10, 25, 50, 100, 250, 500, and 1,000 μM) showed concentration-dependent lethality. Mortality data were collected every 24 h for each group till all the flies were dead. There was no mortality up to the 6th day for health span flies and the 3rd day for late health and transition phase flies. Comparison of survival curves reveals that the response difference among different tested concentrations was significant (log-rank [Mantel–Cox] test. p < 0.0001).

FIGURE 2

Assessing the climbing ability of Drosophila as determined by negative geotaxis assay after exposure to multiple concentrations of rotenone (10, 25, 50, 100, 250, 500, and 1,000 μM). About 500 μM of rotenone-induced clear mobility defects in the adult male fly of early health span on 5th day (A), 25 μM rotenone in late health span fly on the 2nd day (B) and 10 μM rotenone in transition phase fly on the 2nd day (C) and there was no mortality at the selected time points. Observation of mere mobility defects but not mortality of fly is the reason behind selecting above mentioned toxin concentrations and durations of toxin exposure in adult life stage-specific fashion. Hence, these concentrations of toxin and window period of exposure were selected for further studies. Data were collected every 24 h for each group. One-way ANOVA followed by the Newman–Keuls Multiple Comparison Test showed a significant difference in mobility. *p < 0.05, ***p < 0.0001, NS, Not significant.

FIGURE 3

Rotenone inhibits mitochondrial Complex I activity. Upon feeding the early health span fly with 500 μM rotenone for 5 days (A) and 10 μM ROT for 2 days during the transition phase (B), there is a significant decrease of ∼45 and ∼35%, respectively, in the complex I–III activity of the respiratory chain in both the head and body parts of Drosophila melanogaster. Statistical analysis was performed using a t-test (compared to control). *p < 0.05, **p < 0.01.

FIGURE 4

Characterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons. However, rotenone leads to “neuronal dysfunction” as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein. Cartoon showing the position of DAergic neurons in the whole brain of Drosophila melanogaster (A). Brain image of control and induced PD conditions during early health span (B) and transition phase (C). Quantification of DAergic neurons reveals that there is no loss of neuronal number per se in both life stages, early health span (D), and transition phase (E), whereas the quantification of fluorescence intensity (of fluorescently labeled secondary antibodies that target primary antibody anti-TH) reveals a significant decrease in TH protein in both life stages, that is, health stage (F) and transition stage (G). CTR, Control; TD, Treated with rotenone; PAL, Protocerebral anterior lateral; PPL, Protocerebral posterior lateral; PPM, Protocerebral posterior medial. Statistical analysis was performed using a t-test (compared to control). **p < 0.01, ***p < 0.0001, NS, not-significant.

FIGURE 5

Quantification of DA and its metabolites, HVA, and DOPAC using HPLC in fly brain homogenate: Chromatogram of standard DA, HVA, and DOPAC (A) shows specific retention time and chromatogram for fly brain tissue extract (B). The relative level of DA and its metabolites at the selected time points in three stages of the life span, that is, early health span (C); late health span (D); transition phase (E) indicates diminished levels of DA, HVA, and DOPAC illustrating altered DA metabolism in brain-specific fashion in the PD model. Statistical analysis was performed using a t-test (compared to control). *p < 0.05, **p < 0.001.

FIGURE 6

Negative geo-taxis assay of early health phase and transition phase fly in co-feeding regime: Feeding the fly with ROT alone led to a significant reduction in climbing ability which could be significantly improved upon co-feeding with 250 μM curcumin during the early health phase (A) but not during the transition phase (B) of the fly. Climbing ability was assessed on the 5th day for the early health phase fly, while it was assessed on the 2nd day in case of the transition phase. Feeding the fly with curcumin per se did not affect the mobility. One-way ANOVA followed by the Newman–Keuls Multiple Comparison Test showed a significant difference in mobility. ***p < 0.0001; NS, Not significant.