The molecular process regulating the localization of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ) requires agrin, a factor secreted by motor neurons. This process involves various other molecules, but the signaling process in muscles initiated by agrin is poorly understood. We are examining this process in zebrafish which are vertebrates amenable to genetic analysis for the identification of genes important for a biological process and analysis of the in vivo function of these genes. We generated the zebrafish ennui mutation in which AChRs are mislocalized and identified the ennui gene as one encoding for LRP4 that in mammals is required for proper clustering of AChRs. We propose to use the ennui mutants to better understand the in vivo role of LRP4 for the formation of the vertebrate NMJ. We isolated the viable ennui mutation that showed a decreased electrophysiological response at the NMJ. The reduced response was due to a dramatic decrease in synaptic AChRs and high levels of AChRs mislocalized to the ends of muscles. The mutant phenotype is cell autonomous, and exogenous agrin induced AChR clusters in wildtype muscles but not in ennui muscles. These results suggested that the ennui gene encoded for a muscle factor required for agrin-induced localization of AChRs to the NMJ. The ennui gene was identified as lrp4 by a combination of genetic mapping of the mutation and genomic analysis. LRP4 is a member of the low-density lipoprotein receptor family and is expressed by early stage muscles. Although lrp4 was recently found to be critical for proper localization of AChRs in mice, there is little known about how LRP4 may mediate agrin signaling and how it might interact with other well studied components of the agrin-initiated signaling pathway. We propose to explore these issues with experiments that utilize the advantages of zebrafish for examining in vivo gene function.
Aim 1 : We will examine how LRP4 is distributed in muscle by generating antibodies and/or expression of fluorescently labeled LRP4 and see if LRP4 co-localizes with other known NMJ components.
Aim 2 : We will analyze how LRP4 regulates aneural AChR clusters that form prior to innervation by a combination of antisense knockdowns and expression of specific forms of LRP4.
Aim 3 : We will establish whether LRP4 is an aggregation factor and see how LRP4 and MuSK, a component of the agrin receptor complex, are functionally related.
Aim 4 : We will assay how the interaction of LRP4 and MuSK affects the in vivo development of the NMJ.

Public Health Relevance

Zebrafish mutants could serve as animal models for human neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndromes. The phenotype of ennui mutants is reminiscent of the muscle weakness and AChR deficiencies seen in patients afflicted with these diseases. This makes lrp4 a candidate gene for neuromuscular junction disorders in patients of unknown etiology. The fact that ennui mutants are viable makes them more analogous to human disorders and useful for assaying therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS054731-04
Application #
8204505
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Gubitz, Amelie
Project Start
2009-04-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2013-11-30
Support Year
4
Fiscal Year
2012
Total Cost
$325,257
Indirect Cost
$110,882
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Gribble, Katherine D; Walker, Lauren J; Saint-Amant, Louis et al. (2018) The synaptic receptor Lrp4 promotes peripheral nerve regeneration. Nat Commun 9:2389
Ogino, Kazutoyo; Low, Sean E; Yamada, Kenta et al. (2015) RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels. Proc Natl Acad Sci U S A 112:2859-64
Horstick, Eric J; Linsley, Jeremy W; Dowling, James J et al. (2013) Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy. Nat Commun 4:1952
Iwasaki, Kenichi; Taguchi, Meari; Bonkowsky, Joshua L et al. (2013) Expression of arginine vasotocin receptors in the developing zebrafish CNS. Gene Expr Patterns 13:335-42
Dowling, James J; Arbogast, Sandrine; Hur, Junguk et al. (2012) Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy. Brain 135:1115-27
Low, Sean E; Amburgey, Kimberly; Horstick, Eric et al. (2011) TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors. J Neurosci 31:11633-44
Low, Sean E; Zhou, Weibin; Choong, Ingxin et al. (2010) Na(v)1.6a is required for normal activation of motor circuits normally excited by tactile stimulation. Dev Neurobiol 70:508-22
Low, Sean E; Ryan, Joel; Sprague, Shawn M et al. (2010) touche Is required for touch-evoked generator potentials within vertebrate sensory neurons. J Neurosci 30:9359-67
Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori et al. (2009) Defective glycinergic synaptic transmission in zebrafish motility mutants. Front Mol Neurosci 2:26