Neuromuscular effects of G93A-SOD1 expression in zebrafish

Abstract

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis.ResultsWe demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease.ConclusionsThis sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.

Early Development of the Gonadotropin-Releasing Hormone Neuronal Network in Transgenic Zebrafish

“…Fixed embryos were washed with PBS, permeabilized in ice cold acetone for 8 min, rinsed in 0.3% Tween solution in PBS, then blocked for 2 h at room temperature in the dark with a solution of 0.3% Tween and 10% goat serum (in PBS). Following blocking, sections were washed and incubated overnight at 4°C with the primary antibody (anti-SV2, synaptic vesicle protein 2, mouse, 1:200) diluted in the blocking solution (23). Anti-SV2 antibody was purchased from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA, USA).…”

Axonal Transport Defects in a Mitofusin 2 Loss of Function Model of Charcot-Marie-Tooth Disease in Zebrafish

“…They have a well-conserved vertebrate nervous system, and N-ethyl-N-nitrosourea (ENU) induced mutations have so far been generated for approximately 31% of all zebrafish genes (source: Zebrafish Mutation Project http://www.sanger.ac.uk/Projects/D_rerio/zmp/). Furthermore for the study of diseases affecting the nervous system, zebrafish motor and sensory neurons closely resemble those in man, and diseases including spinal muscular atrophy [11], [12], hereditary spastic paraplegia [12], [13] and amyotrophic lateral sclerosis [14], [15] have been successfully modeled using zebrafish.…”

Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS)

“…To determine whether corkscrew-disrupting mutations alleviate axonopathies caused by familial SOD1 mutants in vivo, we conducted experiments using a zebrafish model of ALS (37)(38)(39)(40). We expressed A4V SOD1 with and without the corkscrew-disrupting substitution at Gly33 in a zebrafish TDL6 line in which the primary motor neurons are labeled with GFP and the mitochondria are labeled with dsRed (41).…”

“…To identify which species of SOD1 aggregate is toxic, we monitored the toxicity of various SOD1 mutants as their aggregates (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) harboring single-point substitutions at Gly33 (G33V and G33W) is nontoxic to motor neurons. All peptide segments were prepared identically, and motor neurons were treated with different final concentrations.…”