Trio Regulates Neurite Outgrowth Through A Coupling Mechanism With A Membrane Trafficking Signaling Regulation

Neurite outgrowth and guidance are the fundamental subject in developmental neurobiology.Exploring the molecular mechanisms of these processes is important for understanding the development of our brain and making improvements for treating neuronal disorders.Cytoskeleton dynamics and membrane trafficking processes are both required for proper axon outgrowth and guidance.In developing neurons,cytoskeleton dynamics and membrane trafficking could be regulated by various signaling pathways.However,whether and how these processes are coordinated regulated are not known yet.In this thesis,we proposed a coordination mechanism ofcytoskeleton rearrangement and membrane trafficking through dissecting the functions of Trio in neurite outgrowth.As a key guanine nucleotide exchange factor,Trio contains two GEF domains and several splice variants,conferring the neurite outgrowth with fine orchestrated regulation.We firstly assessed the individual role of the two GEF domains as well as a splicing variant Trio 8 by using mouse lines with neural specific deletion of Trio,Trio GEFD1 and Trio GEFD2.Trio GEFD1 knock-out mice expressed no Trio proteins in the same manner as Trio knock-out mice,and Trio GEFD2 knock-out mice expressed Trio 8 exclusively in cerebellum.We found that Rac1,a small GTPase that could be activated by Trio,did not abolish neurite outgrowth,suggesting that Racl was not responsible for mediating Trio signaling underlying neurite outgrowth.On the other hand,we demonstrated that Cdc42,another Rho family GTPase regulating cytoskeleton organization which could also be activated by GEFD1,was required for the Trio regulated neurite growth since constitutive active Cdc42 could partially rescue neurite outgrowth defect of Trio deficient cerebellar granule neurons.The endogenous Trio8 containing GEFD1 and the upstream region almost completely rescued the neurite outgrowth.This result implies an additional regulatory mechanism within Trio molecule in addition to cytoskeleton-based mechanism of Cdc42 activation.To investigate the possible involvement of membrane mechanism in the context of Trio-regulated neurite outgrowth,we characterized the membrane trafficking defect of Trio-deficient neurites of cerebellar granule neurons.Interestingly,constitutive active Rab8,a Rab family small GTPase regulating membrane trafficking,could also rescue-50%of neurite outgrowth inhibition.Biochemical analysis demonstrated that Trio could facilitate Rab8 activation by interaction with Rab8 GEF Rabin8/Rab3ip.This observation strongly suggests that Trio may regulate neurite growth through double mechanisms regulating both cytoskeleton dynamics and membrane trafficking,and the coupled regulation is conducted through a physic interaction with Rabin8.Our study provides a molecular working model of the coupling regulation of neurite growth.For visualization of neurite outgrowth,we had generated a bacteria artificial chromosome transgenic mice in which a red fluorescent protein was fused to Class ?P-tubulin.mCherry fusion protein was expressed in the nervous system and testis of the transgenic animal,and the fluorescent signal was observed in the neurons that located in the olfactory bulb,cerebral cortex,hippocampal formation,cerebellum,as well as the retina.Besides,Tubb3-mCherry fusion protein mainly distributed in neurites and colocalized with endogenous Class ? ?-tubulin.The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation.Therefore,this transgenic line is a useful tool for scientific community to study neuronal development both in vitro and in vivo.