PtdIns (3,4,5) P3Recruitment Of Myo10is Essential For Axon Development

The polarization of axon and dendrites underlies the unidirectional flow of informationtransfer, which is essential for the establishment of functional circuits in the central nervoussysterm. Remarkble progress was made in identifing polarity proteins in the past threedecades, however, the mechanism of neuronal polarity has not been fully understood. Thecytoskeleton drives neuronal polarization by not only providing structural scaffold forshaping, but also organizing polarized transport of special cargo. Myosin Ⅹ (Myo10), avertebrate-specific unconventional myosin, is not only capable of binding actin andmicrotubule, but also interacting with PtdIns (3,4,5) P3, a lipid product of PI3K which plays akey role in neuronal polarization.In this study, dissociated hippocampal neurons were employed to investigate the role ofMyo10in the axon development. The preferential accumulation of Myo10in axon tip hasbeen revealed in primary culture of hippocampal neurons with the aid of immunofluorescencefrom anti-Myo10antibody in combination with anti-Tuj1antibody as specific marker, whichsuggested the role of Myo10in axon development. Culture neurons were transfected withscrmble miRNA and Myo10miRNA, respectively and analyzed at DIV1,3,5and11.Actin-riched growth cone was less developed after Myo10knock down. The length of longestneurites was significantly shorter than that in the control neurons after24hour culture. Mostneurons expressing Myo10siRNA no longer possessed a single long axon–like neurite,resulting in a loss of the characteristic neuronal polarity. The dynamics and polymerization ofmicrotubule cytoskeleton were disrupted in Myo10-suppressed neurons. In respect thatcytochalasin D rescued the polarity defect caused by Myo10knockdown. It was suggestedthat Myo10was a potential regulator of actin cytoskeleton dynamics in axon development.Moreover, gain-of-function experiment demonstrated that Myo10PHMF induced multipleaxon-like neurites in a motor-independeny manner and PH domain is nessesary for axondevelopment. In order to dissect the molecular mechanism, Myo10PHMF was mutanted inthe second PH domain which was essential for the binding of Myo10to PtdIns (3,4,5) P3according to previous studies. It has been shown that Myo10PHMF (KK/AA) is not capableof accumulat in the tips of axon and lost the ability of inducing multiple axon-like neurites.Consistantly, LY294002, the inhibitor of PI3K disrupted the distribution of Myo10andPHMF could not induce multiple axons under the applycation of LY294002. In addition,Cdc42L28rescued the loss of polarity caused by Myo10miRNA and pull-down assayrevealed that Myo10miRNA suppressed Cdc42activity. In conclusion, Myo10regulatesCdc42activity via recruitment to PtdIns (3,4,5) P3in neuronal polarization. Finally, in vivo studies suggested that Myo10is nessesary for neuronal morphological transition frommultipolar to bipolar during radial neuronal migration.