A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons
Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.
KeywordsTropomyosins Neurite outgrowth inhibition Microfluidic systems NogoA
This work was supported by Project Grant APP1083209 from the Australian National Health and Medical Research Council (NHMRC) (T.F.) and Discovery Project Grant DP180101473 from the Australian Research Council (ARC) (T.F.). We thank Tamara Tomanić (UNSW Sydney) for her constructive feedback and critical reading of the manuscript. This work was performed (in part) at the NSW and South Australian node of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers. M.E.W. would like to acknowledge the support of the Australian Research Council through Discovery Project Grants (DP170103704 and DP180103003) and the National Health and Medical Research Council through the Career Development Fellowship (APP1143377).
TF, MEW and TB supervised the project. TF, HS, AH-B and SF designed the research. HS, AH-B, SF and MB performed the research and analysed the data. TF, HS, AH-B and SF wrote the paper. TF, HS, AH-B, AKS, MB and NT edited the paper. All authors read and approved the final manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial, financial or non-financial relationships that could be construed as a potential conflict of interest.
All procedures were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the University of New South Wales Animal Care and Ethics Committee.
- An H, Brettle M, Lee T, Heng B, Lim CK, Guillemin GJ, Lord MS, Klotzsch E, Geczy CL, Bryant K, Fath T, Tedla N (2016) Soluble LILRA3 promotes neurite outgrowth and synapses formation through high affinity interaction with Nogo 66. J Cell Sci. https://doi.org/10.1242/jcs.182006 CrossRefPubMedGoogle Scholar
- Bonello TT, Janco M, Hook J, Byun A, Appaduray M, Dedova I, Hitchcock-DeGregori S, Hardeman EC, Stehn JR, Bocking T, Gunning PW (2016) A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics. Sci Rep 6:19816. https://doi.org/10.1038/srep19816 CrossRefPubMedPubMedCentralGoogle Scholar
- Brettle M, Patel S, Fath T (2016) Tropomyosins in the healthy and diseased nervous system. Brain Res Bull 126(Pt 3):311–323. https://doi.org/10.1016/j.brainresbull.2016.06.004 CrossRefPubMedGoogle Scholar
- Curthoys NM, Freittag H, Connor A, Desouza M, Brettle M, Poljak A, Hall A, Hardeman E, Schevzov G, Gunning PW, Fath T (2014) Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells. Mol Cell Neurosci 58C:11–21. https://doi.org/10.1016/j.mcn.2013.10.011 CrossRefGoogle Scholar
- Hellman AN, Vahidi B, Kim HJ, Mismar W, Steward O, Jeon NL, Venugopalan V (2010) Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection. Lab Chip 10(16):2083–2092. https://doi.org/10.1039/b927153h CrossRefPubMedPubMedCentralGoogle Scholar
- Li L, Ren L, Liu W, Wang JC, Wang Y, Tu Q, Xu J, Liu R, Zhang Y, Yuan MS, Li T, Wang J (2012) Spatiotemporally controlled and multifactor involved assay of neuronal compartment regeneration after chemical injury in an integrated microfluidics. Anal Chem 84(15):6444–6453. https://doi.org/10.1021/ac3013708 CrossRefPubMedGoogle Scholar
- Schevzov G, Curthoys NM, Gunning PW, Fath T (2012) Functional diversity of actin cytoskeleton in neurons and its regulation by tropomyosin. Int Rev Cell Mol Biol 298:33–94. https://doi.org/10.1016/B978-0-12-394309-5.00002-X CrossRefPubMedGoogle Scholar