Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson's disease

Abstract

Death of dopaminergic (DAergic) neurons in the substantia nigra pars compacta of the human brain is the characteristic pathological feature of Parkinson's disease (PD). On exposure to neurotoxicants, Drosophila too exhibits mobility defects and diminished levels of brain dopamine. In the fly model of sporadic PD, our laboratory has demonstrated that there is no loss of DAergic neuronal number, however, a significant reduction in fluorescence intensity (FI) of secondary antibodies that target the primary antibody-anti-tyrosine hydroxylase (TH). Here, we present a sensitive, economical, and repeatable assay to characterize neurodegeneration based on the quantification of FI of the secondary antibody. As the intensity of fluorescence correlates with the amount of TH synthesis, its reduction under PD conditions denotes the depletion in the TH synthesis, suggesting DAergic neuronal dysfunction. Reduction in TH protein synthesis is further confirmed through Bio-Rad Stain-Free Western Blotting. Quantification of brain DA and its metabolites (DOPAC and HVA) using HPLC-ECD further demonstrated the depleted DA level and altered DA metabolism as evident from enhanced DA turnover rate. Together all these PD marker studies suggest that FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using ZEN 2012 SP2, a licensed software from Carl Zeiss, Germany. This method will be of good use to biologists, as it with few modifications, can also be implemented to characterize the extent of degeneration of different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method using fluorescence microscopy will be a feasible option for fund-constrained neurobiology laboratories in developing countries.

Keywords: Drosophila; dopamine; fluorescence intensity; neurodegeneration; tyrosine hydroxylase.

Figure 1

Assessing the climbing ability of Drosophila by negative-geotaxis assay after exposure to PQ: the climbing speed of flies within 12 s was assayed. Ingestion of 10 mM PQ caused severe mobility defects after 24 (A) and 48 h (B) of exposure. Mobility defects enhanced with exposure duration, but no mortality was observed for the exposure duration of 24 and 48 h. Hence, an exposure window of 24 h was selected for further experiments. Unpaired t-test reveals a significant reduction in mobility of PQ exposed fly compared to control. ***p < 0.001.

Figure 2

Demonstration of DAergic neurons in the whole fly brain: cartoon of Drosophila melanogaster brain illustrating the position of quantifiable DAergic neurons (A) and image of the whole-brain mount of 5 days old male Drosophila captured using ZEN software of Carl Zeiss Fluorescence Microscope using fluorescently labeled secondary antibody targeted against the primary anti-TH antibody (B). In the fly brain, ~140 DAergic neurons (including ~100 neurons of the PAM cluster which cannot be quantified due to the high neuronal density) are arranged in different clusters in each hemisphere. The Scale bar of the brain image in the panel is 20 μm (PAL, Proto-cerebral Anterior Lateral; PAM, Proto-cerebral Anterior Medial; PPL, Proto-cerebral Posterior Lateral; PPM, Proto-cerebral Posterior Medial; VUM, Ventral Unpaired Medial).

Figure 3

Characterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining and quantification of brain TH protein using western blotting: the image depicts the whole brain mount of the adult Drosophila under control and PQ-treated conditions (A) (CTR, Control; TD, PQ-treated). Quantification of DAergic neurons reveals that the neuronal number remains unaffected (B,C), whereas the fluorescence intensity (of fluorescently labeled secondary antibodies that target primary antibody anti-TH) is significantly decreased in all the clusters (D), and in toto (E) between the control and treated group. The scale bar of the brain images in the panel is 20 μm. (CTR, Control; TD, Treated with 10 mM PQ; Represented images are “merged” Z-stacking images; however, the quantification of DA neuronal number and fluorescence intensity is performed in 3D Z-stack images; PAL, Protocerebral anterior lateral; PPL, Protocerebral posterior lateral; PPM, Protocerebral posterior medial). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment in the fly model of PD [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR-control; PQ- paraquat treated)]. Statistical analysis was performed using a t-test (compared to control). *p < 0.05, **p < 0.01, ***p < 0.001; NS, not-significant.

Figure 4

Quantification of DA and its metabolites (DOPAC, HVA) in fly brain tissue extract using HPLC-ECD: the retention time of standard DA, DOPAC, and HVA is shown in the chromatogram (A) and chromatogram for the fly head tissue extract shows the detected catecholamines (B). Quantification of the brain-specific catecholamines revealed that PQ exposure for 24 h depleted brain DA and DOPAC levels, whereas the HVA level is significantly enhanced (C). The result also revealed that in the induced PD condition there is a higher oxidative turnover of DA to its metabolites (DOPAC and HVA) (D). Statistical analysis was performed using an unpaired t-test (compared to the control), *** p < 0.001.