Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Summary

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Abstract

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Introduction

Neurodegenerative diseases affect millions of people each year and the incidence is increasing with an aging population¹. While each neurodegenerative disease has a unique etiology, aggregation of misfolded proteins and breakdown of the proteostasis network are common pathological hallmarks of many of these diseases. Elucidating how disruption of these fundamental and interrelated processes goes awry to contribute to neuronal dysfunction and cell death is critical for understanding neurodegenerative diseases as well as guiding therapeutic interventions. Fluorescence-based imaging allows for investigation of these complex and dynamic processes i....

Protocol

1. Considerations and Preparations for Designing the Image Analysis Experiment

1. Predetermine suitable anatomical, cellular, or subcellular markers to serve as landmarks for standardizing a region of interest (ROI) across different samples, for example 4',6-diamidino-2-phenylindole (DAPI), membrane markers, localized fluorescent protein, etc.
2. Select a fluorescent marker that can delimit discrete puncta beyond background levels for the structure of interest.
   NOTE: This protocol is optimized for proteinaceous structures (e.g., misfolded protein aggregates) and membrane-bound organelles (e.g., autophagy reporter).....

Results

Quantification of the number, area, and intensity of fluorescently tagged mutant Htt aggregates in the Drosophila optic lobe

To investigate misfolded protein aggregation in the central nervous system of a Drosophila model of Huntington's disease, RFP-tagged mutant human Htt with a non-pathological (UAS-RFP-hHttQ15) or pathological expansion (UAS-RFP-hHttQ138) and membrane GFP (UAS-mCD8::GFP

Discussion

The protocol outlined here can be used to robustly and reproducibly quantitate cell biological processes visualized by fluorescence-based imaging. Biological context and technical limitations need to be carefully considered to guide the experimental design. Fluorescent markers of subcellular structures of interest, whether immunohistochemical, dye-based, or genetically expressed, need to be distinguishable above background by morphology and intensity. The UAS/GAL4 system is widely used to drive targeted gene exp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SYLGARD(R) 184 Silicone Elastomer Kit	Dow Corning Corporation	PF184250	Dissection dish
Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish	Corning	351008	Dissection dish
Dumont #5 Forceps	Fine Science Tools	11251-20	Dissection tool
Sodium Chloride	Sigma	S3014	PBS solution
Sodium Phosphate Dibasic	Sigma	S5136	PBS solution
Potassium Phosphate Monobasic	Sigma	P5655	PBS solution
Triton X-100	Sigma	T9284	Washing and antibody incubaton solution.
37% Formaldehyde	VWR	10790-710	Fixation
Disposable Microcentrifuge Tubes (0.5mL, blue)	VWR	89000-022	Fixation, washing, and antibody incubaton.
Plain and Frosted Micro Slides (25×75mm)	VWR	48312-004	Slides for confocal imaging
Micro Cover Glasses, rectangular (22×40mm)	VWR	48393-172	Slides for confocal imaging
Rubber Cement	Slime	1051-A	Mounting
VECTASHIELD Antifade Mounting Medium	Vector Laboratories, Inc.	H-1000	Mounting
Scotch Magic 810 Invisible Tape (19mm×25.4m)	3M Company	810	Mounting
Normal Goat Serum	Thermo Fisher Scientific	PCN5000	Antibody incubaton
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306	Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80°C. Dilute to a working concentration of 10-20 μg/mL in PBTx.
3.5X-90X Stereo Zoom Inspection Industrial Microscope	AmScope	SM-1BNZ	Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator
ImageJ/Fiji	NIH	v1.51u	With SCF_MPI_CBG plugins (version 1.1.2)
FV1000-IX81 Confocal-laser Scanning Microscope	Olympus
Recombinant Construct	Bloomington Drosophila Stock Center	BL37749