Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Summary

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Abstract

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

Introduction

Devastating neurodegenerative disorders such as Parkinson’s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia are increasingly recognized. Unfortunately, most of these neurodegenerative disorders are still without treatments. The widespread clinical use of genome-wide, unbiased genetic tests such as whole exome sequencing has led to an increasing number of genes being implicated in human locomotive disorders. Despite this progress, the pathological progression from early to late stages, remains elusive in these disorders. Drosophila provides one with the genetic tools for studying gene requirement in a controlled spatial and temp....

Protocol

Research on Drosophila melanogaster was in compliance with the University of Alberta’s research guidelines.

1. Fly Collection

1. Collect 20 flies using CO_{2 (g)} anesthetization and place in a 25 mm x 95 mm collection vial containing food.
2. Store vials containing flies horizontally to avoid trapping flies in any liquids that may accumulate in the bottom the vial.
3. Incubate flies for at least 21 hr at 22 °C at 45% humidity in an incubator for approximately for 15 hr. Set the incubator with a 12 hr light : dark cycle.

2. Climbing Assay

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Results

Climbing is a strong and reproducible behavior. Indeed, one day old wild-type flies reach the target distance climbing performance rapidly (25 - 30 sec). Mutant flies present a range of performance from mild (or delayed) to complete inability to climb to the target. We illustrate this here with two different mutant alleles. The first one is a severe allele of the gene spastin caused by a complete deletion of the spastin gene (spas^5.75) ¹⁸. In this line (spas^5.75 with TM6b) one day old fli.......

Discussion

Drosophila has already proven to be an excellent model in Parkinson’s disease¹⁴ and other neurodegenerative conditions^1,2. In addition to the genetic tools available in Drosophila, its genome is highly conserved for genes involved in neurological disorders¹⁹. The advent of genome wide genetic screening methods (including whole exome sequencing) is likely to continue to provide a larger list of candidate genes associated with human movement disorders. The developmen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila stocks			The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.
Video camera			Any digital camcorder will do. Make sure they can focus on close object.
Graduated cylinder	Kimble	20028W	Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.
Computer			Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point.