A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Summary

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

Abstract

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

Introduction

Neuronal degeneration occurs during normal development and can be caused by the natural aging process, injury, stress, or disease states. Drosophila melanogaster, the common fruit fly, provides a simple and powerful model organism to study neurodegeneration due to the remarkable similarities in the molecular mechanisms that drive the degeneration of neurons. These similarities are highlighted by the fact that approximately 70% of disease-associated human genes have a Drosophila homolog.¹ Additionally, numerous assays and technological tools to study human neurodegenerative diseases have been developed and utilized in Droso....

Protocol

1. Preparation of Reagents and Equipment

1. Prepare 1x Dissection Buffer (70 mM NaCl, 5 mM KCl, 0.02 mM CaCl₂, 20 mM MgCl₂, 10 mM NaHCO₃, 115 mM sucrose, 5 mM trehalose, 5 mM HEPES; pH 7.2).
2. Prepare 1x Phosphate-Buffered Saline (PBS).
3. Prepare 1x PBT using 1x PBS with 0.01% Triton X-100.
4. Prepare fruit juice agar plates.¹⁴ Briefly, mix 30 g agar in 700 mL of H₂O and autoclave. Dissolve 0.5 g of methyl paraben in 10 mL of ethanol and add solution to 300 mL of fruit juice concentrate (grape or apple). Mix concentrate into autoclaved agar solution and pour into t....

Results

Using the procedure presented here, we have demonstrated that mechanical neuronal injury allows for temporal dissection of neurodegenerative events.^14,18 The sequence of events has been previously characterized and begins with an immediate disruption of the cytoskeleton, followed by axonal trafficking defects, an accumulation of ubiquitinated proteins, and subsequent neurodegeneration 24 h post-injury. Prior to injury, WT NMJs sho.......

Discussion

The neuronal mechanical injury described earlier and demonstrated here can be used to induce injury/stress in the segmental nerves of Drosophila larvae.^4,5,6,14 This experimental technique has been employed previously to dissect the temporal sequence of events leading to neurodegeneration, as well as to examine transcriptional changes in the motor neuron cell bodies post injury.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micro-dissecting scissors	Fine Science Tools	15000-08
Dumont #3 Forceps	Fine Science Tools	11231-30	Some people prefer size 3, while others prefer size 5
Dumont #5 Forceps	Fine Science Tools	11251-30	Some people prefer size 3, while others prefer size 5
CO2 Air Tank	Tech Air	UN 1013	Various tank sizes can be purchased
CO2 Anesthetizing Apparatus	Genesee Scientific	59-114
Stainless-steel pins, size 0.1	Fine Science Tools	26002-10
SylGard 184 Silicone Elastomer, Base and Curing Agent	Dow Corning	3097358-1004	To pour dissecting plates
Bouin's Solution	Sigma	HT 10132-1L	Antibodies should be tested for their efficiency in Bouin's and PFA
4% Paraformaldehyde (PFA) in Phosphate-Buffered Saline (PBS)	Affymetrix	FLY-8030-20	Antibodies should be tested for their efficiency in Bouin's and PFA
Dissecting Stereo MIcroscope	AmScope	SM-1BZ
Light Source	AmScope	HL150-AY-220V
anti- nc82 antibody	Developmental Studies Hybridoma Bank	nc82-s
anti-discs large antibody	Developmental Studies Hybridoma Bank	AF3
Alexa Fluor anti-horseradish peroxidase	Jackson Immunoresearch	123-545-021; 123-585-021; 123-605-021	One can you Alexa Fluor® 488, 594 or 647
Flystuff Grape Juice Agar Premix	Genesee Scientific	47-102
Microscope slides	Genesee Scientific	29-101
Glass Coverslips	Fisher Scientific	12-545-87
Thermo Scientific Nalgene Utility Box	Fisher Scientific	03-484C	Used to create humid chamber for larval recovery