Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, p75NTR and Rho-A.
The presence of multiple axon growth inhibitors may partly explain why central nervous system axons are generally incapable of regenerating after injury. Using RNA interference (RNAi) in dorsal root ganglia neurons (DRGN), we demonstrate siRNA-mediated silencing of components of the inhibitory signalling cascade, including p75NTR, NgR and Rho-A mRNA, of 70%, 100% and 100% of the relevant protein, respectively, while changes in neither protein levels nor cellular immunoreactivity were detected using the relevant scrambled siRNA control sequences. Importantly, after 48 h in culture after siRNA-mediated knockdown of Rho-A, neurite outgrowth was enhanced by 30% compared to that after p75NTR and 50% after NgR silencing. By 3 days, a 5-, 3.5- and 6.5-fold increase in betaIII-tubulin protein levels were observed compared to controls without siRNA after knockdown of p75NTR, NgR and Rho-A, respectively. Together, these results suggest that Rho-A knockdown might be the most effective target for a disinhibition strategy to promote CNS axon regeneration in vivo.
Ahmed
Zubair
Z
Molecular Neuroscience Group, Department of Medicine, University of Birmingham, 3rd Floor Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, UK. z.ahmed.1@bham.ac.uk
Dent
Russell G
RG
Suggate
Ellen L
EL
Barrett
Lee B
LB
Seabright
Ruth J
RJ
Berry
Martin
M
Logan
Ann
A
D000818:Animals; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D025461:Feedback, Physiological / Q000187:drug effects / Q000502:physiology; D058851:GPI-Linked Proteins; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009185:Myelin Proteins / Q000378:metabolism / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000494:pharmacology / Q000502:physiology*; D009416:Nerve Regeneration / Q000187:drug effects / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000378:metabolism*; D000070816:Nogo Receptor 1; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020800:Receptor, Nerve Growth Factor; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics* / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism
D058851:GPI-Linked Proteins; D009185:Myelin Proteins; D009414:Nerve Growth Factors; D000070816:Nogo Receptor 1; D034741:RNA, Small Interfering; D020800:Receptor, Nerve Growth Factor; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D018000:Receptors, Peptide; C486581:Rtn4r protein, rat; D014404:Tubulin; D020742:rhoA GTP-Binding Protein