CRMP2 And CRMP4 Regulate The Axonal Development Of Hippocampal Neurons

Objectiveand significanceNeurons are polarized by microtubule and microfilaments movements to form dendrites and axons through the growth cone to sense extracellular growth and orientation signals.And then,thousands hundred million neurons that connect together by many synapses form an accurate and complex neural network.The communication and interaction between microtubules and microfilaments are considered as the main cause of cell polarization.However,which proteins and how these proteins mediate their interaction are still unknown.Collapsin response mediator proteins(CRMPs),consisting of five cytosolic proteins(CRMP 1-5),are highly expressed in the developing nervous system,mediating growth cone guidance,neuronal polarity,and axon and dendrite elongation.CRMPs likely exist as homo-or heterotetramersin vivo.They can interact with microtubules and actin respectively.How can CRMPs associate with microtubules and actin coordinated cytoskeletal dynamics? The current study aimed to investigate the role of CRMPs coordinated cytoskeleton dynamics in neuronal growth cone development and axonal elongation.Investigating regulation of CRMPs coordinated cytoskeleton dynamics in neuronal growth cone development and axonal elongation conduces to understand the cause of nervous developmental disorders,and then leads us to have a chance to find a new route and therapy target.Materials and methodsWe first constructed GST-CRMPs and purified the proteins.We extracted growth cone lysates to perform GST-pulldown to confirm the interaction between CRMPs and tubulin/actin.By the application of Co-IP using CRMPs/tubulin/actin antibodies,we tried to observe their interaction in vivo,and by immunofluorescence to reveal their co-localization in growth cones.By using CRMPs antibodies in Co-IP and immunocytochemistry,we tried to confirm the interaction between CRMPs and their colocalizaiton in growth cones.We designed CRMPs siRNA fragments to confirm the role of CRMPs in axon development.And by overexpression of truncated CRMPs which cannot bind to tubulin or actin,we tried to demonstrate whether CRMPs mediated cytoskeleton dynamics would affect the development of growth cones and axons.Results 1.The results of GST-pulldown showed that CRMP1-5 showed nearly equal abilities to interact with tubulin and actin,except CRMP3 had less affinity for actin.By contrast,the GST control group showed no interaction with either tubulin or actin.And CRMP2 and CRMP4 interact with the cytoskeleton in vitro.2.CRMP2 and CRMP4 mainly distributed in the central(C)zone and the transition(T)zone,with an especially strong signal in the transition(T)zone of the growth cone.In addition,CRMP2 and CRMP4 colocalized with microtubules and actin in the T zone.3.The results of Co-IP and GST-pulldown showed that CRMP2 interacted with CRMP4.In addition,truncated GST-CRMP2ΔC322 which could not interact with tubulin can also interact with CRMP4,and truncated GST-CRMP4ΔC471 which could not interact with actin can also interact with CRMP2.By applying immunocytochemistry methods,we detected CRMP2 and CRMP4 colocalized in the growth cones.4.By the application of our designed siRNA fragments,the level of CRMP4 but not CRMP2 was significantly suppressed.In hippocampal neurons,the endogenous level of CRMP4 was also markedly silenced as shown by immunostaining.The knockdown of CRMP4 dramatically reduced the size of the growth cones in hippocampal neurons at DIV 3,and co-silencing with CRMP2 resulted in further growth cone shrinkage.Overexpression of CRMP2 rescued the inhibitory effect of the CRMP4 knockdown on growth cone development.Moreover,knockdown of CRMP4 impaired axonal growth,and co-silencing of CRMP2 caused further impairment.Overexpression of CRMP2 also partially rescued the inhibitory effect of CRMP4 siRNA on axonal growth.Overexpression of CRMP4 markedly enlarged the size of the growth cones,however,when CRMP4 was co-transfected with the CRMP2 siRNA fragment,the size of the growth cones was not different from that of controls.Overexpression of CRMP4 promoted axonal growth,while co-transfection of the siCRMP2 fragment blocked the promoting effect of CRMP4 on axonal growth.5.Although CRMP4ΔC471 could not interact with actinand CRMP2ΔC322could not interact with tubulin,they did not affect the interaciton of CRMP2 and CRMP4.CRMP4ΔC471 significantly reduced the growth cone size when co-transfected with CRMP2,and CRMP2ΔC322 suppressed the promoting effect of CRMP4 overexpression on growth cone development.A similar result for axonal elongation: CRMP4ΔC471 reversed the promoting effect of CRMP2,and CRMP2ΔC322 reversed the promoting effect of CRMP4 on hippocampal axonal elongation.Conclusion 1.CRMPs interact with tubulin and actinin vivo and in vitro.2.CRMP2 and CRMP4 coexist in the growth cone of neurons and coexist in the T zone.3.Both CRMP2 and CRMP4 can promote axonal growth,and they may form a complex to function collaboratively.4.The complexes of CRMP2 and CRMP4 work coordinately,via the interaction with tubulin and actin,to regulate growth cone development and axon elongation.