dissection-immunohistochemistry-drosophila-adult-leg-to-detect

Dissection and Immunohistochemistry of the <em>Drosophila</em> Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Rhode Island College

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

Mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS) in humans. ALS is a neurodegenerative disease characterized by progressive motor neuron loss leading to paralysis and inevitable death in affected individuals. Using a gene replacement strategy to introduce disease mutations into the orthologous  () gene, here, we characterize changes at the neuromuscular junction using longer-lived  mutant adults. Homozygous  or  flies display progressive walking defects with paralysis of the third metathoracic leg. In dissected legs, we assessed age-dependent changes in a single identified motor neuron (MN-I2) innervating the tibia levitator muscle. At adult eclosion, MN-I2 of  or  flies is patterned similar to wild-type flies indicating no readily apparent developmental defects. Over the course of 10 days post-eclosion, MN-I2 shows an overall reduction in arborization with bouton swelling and loss of the post-synaptic marker  () in mutant  adults. In addition, increases in polyubiquitinated proteins correlate with the timing and extent of MN-I2 changes. Because similar phenotypes are observed between flies homozygous for either  or  alleles, we conclude these NMJ changes are mainly associated with  loss-of-function. Together these studies characterize age-related morphological and molecular changes associated with axonal retraction in a  model of ALS that recapitulate an important aspect of the human disease.This article has an associated First Person interview with the first author of the paper.

Geoff Stilwell

Department of Biology

Rhode Island College

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Geoff Stilwell

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Rhode Island College

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Department of Biology