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The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction

Abstract

TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. We employed Drosophila melanogaster to investigate the function of the closely related gene, Tmep (CG12004), at the neuromuscular junction. We show that Tmep is required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many postsynaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Additional branches and satellite boutons at Tmep mutant neuromuscular junctions are consistent with an activity-dependent synaptic overgrowth. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, we show that Tmep is required in motor neurons for proper larval locomotion and excitability, and that its reduction increases levels of presynaptic calcium. Locomotor defects can be rescued by presynaptic knockdown of endoplasmic reticulum calcium channels or by reducing evoked release probability, further suggesting that excess synaptic activity drives behavioral deficiencies. Our work establishes a critical function for Tmep in the regulation of synaptic transmission and locomotor behavior.

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Data Availability

Flies and plasmids created during this study will be available from the authors or deposited at the Bloomington Drosophila Stock Center (BDSC) or at Addgene.

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Acknowledgements

The authors would like to thank all members of the Bhattacharya lab for thoughtful comments on the manuscript. We also thank Dr. Konrad Zinsmaier for sharing his electrophysiology equipment as well as his expertise for our work. We thank Drs. Kimberly Chapman and Seth Berger at Children’s National Medical Center for communicating unpublished information. We thank the York Protein Production Facility, the York Imaging and Cytometry Facility, and the University of Arizona Marley Microscopy Core Facility for their assistance.

Code Availability

All code used in image analysis is deposited on our research group’s GitHub page and is freely available to all (https://github.com/martharcb).

Funding

This work was supported by R01NS105680, a Muscular Dystrophy Association Development grant, and the Arizona Technology and Research Initiative Fund (TRIF) (to M.R.C.B.), the MRC (UK) grant MR/M013596/1 to S.T.S. and a Biotechnology and Biological Sciences Research Council Studentship grant BB/M011151/1 (to S.T.S. and E.B.).

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S.T.S. and M.R.C.B. proposed the research and assisted in experimental design. T.S.C., E.B., and N.E.K. designed and performed experiments. All three first authors (indicated with *) made significant discoveries leading to this manuscript, resulting in all three being listed as equal first authors. T.S.C. and M.R.C.B. wrote the manuscript, with editing by E.B. and S.T.S.

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Correspondence to Martha R. C. Bhattacharya.

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Tiffany S. Cho, Eglė Beigaitė and Nathaniel E. Klein contributed equally to this work.

Supplementary Information

Online Resource 1

Tmep is expressed broadly throughout the larvae. RedStinger:NLS (red and orange) shows nuclei of cells in which endogenous Tmep is expressed. a, Ventral nerve cord. Green is Repo (glial nuclei). b, Neuromuscular junction. Green is discs large (DLG). c, Gut expression. d, Fat body expression. For all figures, scale bars = 20 μm. (PNG 1297 kb)

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Cho, T.S., Beigaitė, E., Klein, N.E. et al. The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction. Mol Neurobiol 59, 2605–2619 (2022). https://doi.org/10.1007/s12035-022-02760-3

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