Motor Neuron Abnormalities Correlate with Impaired Movement in Zebrafish that Express Mutant Superoxide Dismutase 1

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

[1]  L. Petrucelli,et al.  A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism , 2018, Acta Neuropathologica.

[2]  Adam J. Svahn,et al.  Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy , 2017, The Journal of Neuroscience.

[3]  O. Hermine,et al.  ALS Clinical Trials Review: 20 Years of Failure. Are We Any Closer to Registering a New Treatment? , 2017, Front. Aging Neurosci..

[4]  G. Nicholson,et al.  A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous System and Neurodegenerative Disease. , 2017, Zebrafish.

[5]  Robert H. Brown,et al.  Decoding ALS: from genes to mechanism , 2016, Nature.

[6]  T. Ramesh,et al.  ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis , 2016, Molecular Neurodegeneration.

[7]  T. Becker,et al.  Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages. , 2016, Human molecular genetics.

[8]  H. Horvitz,et al.  Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice , 2015, Neuron.

[9]  L. Petrucelli,et al.  C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD , 2015, Neuron.

[10]  Kevin F. Bieniek,et al.  C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits , 2015, Science.

[11]  Cassie S. Mitchell,et al.  State of the field: An informatics-based systematic review of the SOD1-G93A amyotrophic lateral sclerosis transgenic mouse model , 2015, Amyotrophic lateral sclerosis & frontotemporal degeneration.

[12]  Michael Q. Zhang,et al.  Activity-dependent FUS dysregulation disrupts synaptic homeostasis , 2014, Proceedings of the National Academy of Sciences.

[13]  C. Haass,et al.  Genomic editing opens new avenues for zebrafish as a model for neurodegeneration , 2013, Journal of neurochemistry.

[14]  A. Higginbottom,et al.  A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening , 2013, Disease Models & Mechanisms.

[15]  Ralf Mikut,et al.  Identification of Nonvisual Photomotor Response Cells in the Vertebrate Hindbrain , 2013, The Journal of Neuroscience.

[16]  Gene W. Yeo,et al.  ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43 , 2013, Proceedings of the National Academy of Sciences.

[17]  T. Hortobágyi,et al.  Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion , 2012, Acta Neuropathologica.

[18]  J. Dowling,et al.  Neuromuscular effects of G93A-SOD1 expression in zebrafish , 2012, Molecular Neurodegeneration.

[19]  A. Goris,et al.  EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans , 2012, Nature Medicine.

[20]  S. Rivest,et al.  Sensorimotor and cognitive functions in a SOD1 G37R transgenic mouse model of amyotrophic lateral sclerosis , 2011, Behavioural Brain Research.

[21]  L. Petrucelli,et al.  Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice , 2011, Molecular Neurodegeneration.

[22]  Bruce L. Miller,et al.  Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS , 2011, Neuron.

[23]  David Heckerman,et al.  A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD , 2011, Neuron.

[24]  E. Brustein,et al.  FUS and TARDBP but Not SOD1 Interact in Genetic Models of Amyotrophic Lateral Sclerosis , 2011, PLoS genetics.

[25]  Thomas S. Becker,et al.  Zebrafish: An integrative system for neurogenomics and neurosciences , 2011, Progress in Neurobiology.

[26]  C. Beattie,et al.  A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease , 2010, Disease Models & Mechanisms.

[27]  E. Brustein,et al.  In the swim of things: recent insights to neurogenetic disorders from zebrafish. , 2010, Trends in genetics : TIG.

[28]  Laurent Ségalat,et al.  High‐throughput screening and small animal models, where are we? , 2010, British journal of pharmacology.

[29]  G. Rouleau,et al.  Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo. , 2010, Human molecular genetics.

[30]  Christian Laggner,et al.  Rapid behavior—based identification of neuroactive small molecules in the zebrafish , 2009, Nature chemical biology.

[31]  N. Cairns,et al.  TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration , 2009, Proceedings of the National Academy of Sciences.

[32]  R. Miller,et al.  Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND) , 2003, Amyotrophic lateral sclerosis and other motor neuron disorders : official publication of the World Federation of Neurology, Research Group on Motor Neuron Diseases.

[33]  R. Köster,et al.  Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio). , 2009, Brain : a journal of neurology.

[34]  Xun Hu,et al.  Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6 , 2009, Science.

[35]  J L Haines,et al.  Supporting Online Material Materials and Methods Figs. S1 to S7 Tables S1 to S4 References Mutations in the Fus/tls Gene on Chromosome 16 Cause Familial Amyotrophic Lateral Sclerosis , 2022 .

[36]  Xun Hu,et al.  TDP-43 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis , 2008, Science.

[37]  P. Carmeliet,et al.  Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish. , 2007, Human molecular genetics.

[38]  N. Hukriede,et al.  Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene , 2007, Nucleic acids research.

[39]  P. Andersen,et al.  Motor Neuron Disease in Mice Expressing the Wild Type-Like D90A Mutant Superoxide Dismutase-1 , 2006, Journal of neuropathology and experimental neurology.

[40]  U. Monani,et al.  Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding , 2003, The Journal of cell biology.

[41]  E. Brustein,et al.  Development of the locomotor network in zebrafish , 2002, Progress in Neurobiology.

[42]  J. Dowling,et al.  Small molecule developmental screens reveal the logic and timing of vertebrate development. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[43]  M. Gurney,et al.  Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. , 1994, Science.

[44]  J. Haines,et al.  Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis , 1993, Nature.

[45]  M. Molloy,et al.  Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype , 2017, Human molecular genetics.

[46]  W. Robberecht,et al.  Modeling neurodegenerative diseases in zebrafish embryos. , 2011, Methods in molecular biology.

[47]  Jonathan J. Sager,et al.  Transgenic zebrafish models of neurodegenerative diseases , 2009, Brain Structure and Function.

[48]  R. Miller,et al.  Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). , 2003, Amyotrophic lateral sclerosis and other motor neuron disorders : official publication of the World Federation of Neurology, Research Group on Motor Neuron Diseases.