Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters

Abstract

Background: Studies of mitochondrial morphology vary in techniques. Most use one morphological parameter while others describe mitochondria qualitatively. Because mitochondria are so dynamic, a single parameter does not capture the true state of the network and may lead to erroneous conclusions. Thus, a gestalt method of analysis is warranted.

New method: This work describes a method combining immunofluorescence assays with computerized image analysis to measure the mitochondrial morphology within neuritic projections of a specific population of neurons. Six parameters of mitochondrial morphology were examined utilizing ImageJ to analyze colocalized signals.

Results: Using primary neuronal cultures from Drosophila, we tested mitochondrial morphology in neurites of dopaminergic (DA) neurons. We validate our model using mutants with known defects in mitochondrial morphology. Furthermore, we show a difference in mitochondrial morphology between cells treated as control or with a neurotoxin inducing PD (Parkinson's Disease in humans)-like pathology. We also show interactions between morphological parameters and experimental treatment.

Comparison with existing methods: Our method is a significant improvement of previously described methods. Six morphometric parameters are quantified, providing a gestalt analysis of mitochondrial morphology. Also it can target specific populations of mitochondria using immunofluorescence assay and image analysis.

Conclusions: We found that our method adequately detects differences in mitochondrial morphology between treatment groups. We conclude that some parameters may be unique to a mutation or a disease state, and the relationship between parameters is altered by experimental treatment. We suggest at least four variables should be considered when using mitochondrial structure as an experimental endpoint.

Keywords: Drosophila; Mitochondria; Parkinson; drp1; opa1.

Figure 1. Visualizing mitochondria and quantifying their morphology in a specific cell type

Example images from immunocytochemistry method of quantifying mitochondria in neurites of dopaminergic (DA) neurons. A) DA neurons (green) are identified by antibody to tyrosine hydroxylase (anti-TH). B) Mitochondria (red) are identified by MitoTracker staining. C) Colocalized signals (white) from anti-TH and MitoTracker show as white and represent mitochondria specifically from DA soma and neurites. D) Threshold painting of the mitochondrial signal. The soma is indicated by a yellow arrow. The grey line indicates the selected area to be analyzed (soma excluded). E) Resulting analyzed image from D. Soma and particles outside of specified size range are excluded. Outlines of remaining mitochondrial signals are drawn in black. Image taken at 7 days in vitro (DIV), scale bar = 20 μm.

Figure 2. Mitochondrial morphology in fission and fusion mutants

Quantification of mitochondrial morphology in control and mutant neuronal cultures. Top row: dopaminergic (DA) cells for control, drp1 mutants, and opa1 mutants. Signal is from antibody to tyrosine hydroxylase (TH), selectively marking soma and neurites of DA neuron. Middle row: images of MitoTracker staining for control, drp1 and opa1. Mitochondria from all cell types are stained. Bottom row: overlapped image of anti-TH and MitoTracker signals. Colocalized signals are shown in white, i.e. are mitochondria from DA neurons. Single-embryo cell cultures on individual glass coverslips were prepared from drp1/CyO-GFP and opa1/CyO-GFP fly lines. At 3 DIV, coverslips were selected by the presence/intensity of the live-GFP signal. Coverslips without a live-GFP signal were homozygous for drp1 or opa1. Coverslips with the brightest live-GFP signals were used as controls. At 7 DIV, coverslips were stained with anti-TH and MitoTracker and images were taken for analysis (scale bar = 20 μm, insets are of areas outlined in blue).

Figure 3. Cells with decreased mitochondrial fission or fusion have changes in all measured parameters

Quantification of mitochondrial morphology in control and mutant neuronal cultures. Graphs showing morphological characteristics of mitochondria: number, size, interconnectivity, and elongation. drp1 mutants had larger, more interconnected, and elongated mitochondria compared to control and opa1, while opa1 had smaller, less interconnected, and less elongated mitochondria compared to drp1 and control. Both drp1 and opa1 had fewer mitochondria than control – likely because in drp1 cells, the mitochondria are all connected and thus not counted as separate, and in opa1 cells because the fragmented mitochondria are translocated to the soma where they are not included in analysis. At 7 DIV, images were taken for analysis (Tukey's HSD test; number of experiments: Control, n = 7; drp1, n = 3; opa1 n = 3; *** p < 0.001).

Figure 4. MPP⁺ treatment causes fragmented mitochondrial morphology

Primary neuronal cell cultures were prepared from wild-type fly embryos. At 3 DIV, cultures were treated with 40 μM MPP⁺, or as control. At 7DIV, cells were stained with anti-TH and MitoTracker for the colocalization analysis of mitochondria in DA neurons. A) Example images of control and MPP⁺-treated dopaminergic (DA) neurons from immunocytochemistry colocalization method (anti-TH and MitoTracker). Mitochondria of dopaminergic neurons are shown in white (scale bar = 20 μm, insets are of areas outlined in blue). B) All four parameters showed a significant decrease from control when treated with MPP⁺. Primary neuronal cell cultures were prepared from wild-type fly embryos. At 3 DIV, cultures were treated with 40 μM MPP⁺, or as control. At 7DIV, cells were stained with MitoTracker and anti-TH for the colocalization analysis, and images were taken for analysis (Student's t-test; *** p < 0.001); number of DA neurons analyzed: Control = 29, MPP⁺ = 51).

Figure 5. Parameters of mitochondrial morphology correlate with specific treatment groups

Principal component analysis was used to compare the relationships of the 6 mitochondrial morphology parameters in each of the 4 treatment groups. The different treatment groups had a center of gravity in separate quadrants, except for the MPP⁺ and opa1 treatments, which clustered together - expected as both had similar phenotypes of mitochondrial morphology. The control group appeared to have opposite correlations with the parameters compared to the MPP⁺ and opa1 treatments, while the drp1 treatment differed from all three of the other groups. Individuals factor map shows squares for the center of gravity for each of the 4 treatment groups surrounded by 95% confidence ellipses (CONT n = 52, drp1 n = 26, opa1 n = 64, MPP⁺ n = 51).