Regulation Of Rho Kinase To Neurite Growth And Microtubule Rearrangement In Cultured Hippocampal Neurons Of Rats

Objective and significance:Nervous system,a highly ordered and sophisticated network,is composed of thousands hundred million neurons that connect together by many perfect synapses. A dynamic process during neuronal development involving differentiation,migration, polarization and guidance growth is essential for establishing precise synaptic connections with their target cells.Growth and guidance cues are detected by the specialized tips of growing neurites,termed the growth cones.Growth cones have sensory and motor capabilities enabling them to integrate the information conveyed by these cues into appropriate behavior of microtubule and microfilament to form sprouting axons and dentrites or to be collapsed.The rearrangement of cytoskeleton is the key to neurite growth,guidance and migration,and Rho GTPases is the important cue regulating motor behavior of cytoskeleton.Extracellular growth and guidance factors engaged with surface membrane receptors leads to intracellular signaling cascades including Rho kinase and CRMPs,whose final common path of action is principally the cytoskeleton.Assembly and disassembly of microtubule or microfilament result neurites in sprouting or being collapsed.During development of the nervous system,the complicated structure and precise network they form are the basis of the nervous function.Neuronal development is not only a process from young to maturation,but also involves neuronal reparation and regeneration,which is considered to be a reappearance of neurite growth after nervous tissue damage. Investigating regulation of Rho kinase to neurite growth and microtubule rearrangement conduces to understanding the nervous regeneration after damage and cause of nervous degenerative diseases,and then leads us to have a chance to find a new route and therapy target.Materials and methods:Total RNA was extracted from rat hippocampus in different development stages. Growth and guidance factors in addition to Rho kinase signaling molecules were detected by RT-PCR including GAP-43,Nogo-A,netrin-1,Sema 3A,Nrp-1,Rho-A, Rac-1,CRMP-1 and Tubβ3 mRNA.And then,cultured rat hippocampal neurons treated with LPA and Y27632 at dose of 200ng/ml,accelerator and inhibitor of Rho kinase,were utilized to make neurite extension and microtubule rearrangement be observed.Neurites extension was analyzed by atomic force microscopy and a quantification assay kit,meanwhile,the images of microtubules in immunofluorescence stained neurons were obtained by using a confocal laser microscopy system.The subcellular localization and distribution of vinculin protein were observed in cultured neurons.A CRMP-1-PGEM-T Easy recombinant was constructed to detect what CRMP-1 protein influences neurite growth.The undirected cloning was employed to construct CRMP-1-phrGFPⅡ-N eukaryotic expression vector which was conveyed into PC12 cells induced by nerve growth factor by transfection of positively charged liposome.Results:(1)The expression of growth and guidance factors and Rho kinase signaling molecules mRNA in development of rat hippocampus: During development of rat hippocampus,transcription of GAP-43 gene was detected to be highest in embryonic rats,and significantly lower in neonate,juvenile and adult rats(P＜0.05),however,higher expression was still found in senile rats. Nogo-A was mainly expressed in embryonal stage,then decreased just like GAP-43. The higher expression was detected in adult and senile rats.Netrin-1 gene was mainly transcripted in embryonic,neonate and adult rats,compared with juvenile and senile rats.Transcription of Sema 3A gene was step down with hippocampal development, however,its receptor Nrp-1 mRNA was gradually increased from juvenile to senile rats.Rho-A and Rac-1,signaling molecules,shared similar expressed pattern that demonstrated an evident phase expression with hippocampal development.The higher expression was in embryonic,juvenile and senile rats,otherwise,lower in neonate and adult rats.Expressed quantity of Rac-1 mRNA always super to Rho-A, when two signaling molecules were compared.CRMP-1 and tubulin gene possessed a similar pattern in transcription from embryos to adults,higher in embryonic,neonate and juvenile rats.In senile rats,transcription of CRMP-1 gene was significantly increased(P＜0.05),while tubulin stayed in the same level compared with adult rats.(2)The regulation of Rho kinase to neurite growth and microtubule rearrangement in cultured hippoampal neurons of rats:Numbers of neurites in cultured neurons treated with LPA were decreased and some neurites were only shown rudimental roots observed with atomic force microscopy.The number of 1st-3th order neurites were decreased significantly compared with the control cells(P＜0.05)and the shorter rudimental roots were only observed.Treated with Y27632 after LPA incubation,cellular neurites changed markedly.The important difference from control cells is that more 1st neurites accompanying radiatiform 2nd neurites sprouted from the cell body.Their numbers were increased significantly(P＜0.05).Optical density of neurites extraction was decreased after treated with LPA compared with the control cells(P＜0.05),while it was contrast to LPA treated cells after incubation with Y27632.The neurites outgrowth was super to the control cells but no significant difference.Visible microtubules were viewed in cell bodies,neurites and growth cones in cultured hippoampal neurons analyzed by confocal laser microscopy system.LPA treated neurons exhibited deficient or short neurites which were stained superficial fluorescence in distal of them.There were no ramified and bird nest like microtubules in cell bodies substituted by irregular microtubules which had different diameters and arranged untidily.In neurites the arrangement of microtubules just liked a braid, where comparatively large crevices were observed among fasciculated microtubules. The intrinsical fan-shaped growth cones were to be missing and just left a tiny tips at the end of neurites where microtubules were almost disappeared.Y27632 treated neurons recuperated more and more visible ramified and bird nest like microtubules in their bodies.These microtubules shared similar diameters and circled around nucleus under the cell membranes.Some microtubules with dissociative ends extended to the membranes and grew into neurites accompanying their branches. Meanwhile,microtubules in neurites were still irregular and had some crevices but better than in LPA treated cells.Ends of neurites kept growth cones to be untypical fan-shaped where visible straight and snaked microtubules extended into the inner of growth cones.The longer could reach the centre region of growth cones and the shorter the basal region of growth cones.Vinculin protein shown green fluorescence were scattered on the membranes in control and LPA treated neurons.After treated with Y27632 vinculin protein aggregated together and concentrated at the edge of cells and the basal of neurites.(3)Construction of CRMP-1-phrGFPⅡ-N eukaryotic expression vector and regulation to neurites outgrowth: Total mRNA was extracted from rat hippocampus and amplification of objective gene segment about 1800bp was consistent with expectation for CRMP-1.After connection and transformation white clone was selected.The recombinant cut by EcoR I included 1800bp objective and 3000bp cloning vector gene segments, otherwise,the size of recombinant not cut by EcoR I was about 4800bp.Two-way sequencing results demonstrated that 1746bp objective gene segment was according with CRMP-1 gene size and sequence in Genbank.The obtained sequence of CRMP-1 generated some mutation in four differently localized bases.However, 99.8%bases of the objective gene were still homologous to sequence in Genbank, and the obtained sequence was able to translate correct protein.After CRMP-1 gene segment was connected with expression vector,single positive direction clone was harvested and the identified size of CRMP-1-phrGFPⅡ-N recombinant was 6700bp shown on the electrophoresis strip.Two segments of 4900bp and 1800bp were obstained after the recombinant was cut by EcoR I.For transfection,PC12 cells were induced by nerve growth factor at dose of 50ng to differentiate them to be neuron.In control cells transfected with not including CRMP-1 gene vector,green fluorescence was detected in cell body and longer neuritis.After transfected with CRMP-1-phrGFPⅡ-N vector,distribution of fluorescence was similar to the control cells.The main difference was focused on the neurites that were shorter than the control cells and longer neurites super to two times cell body diameter were almost not found.Almost all of green fluorescence cells show some shorter neurites than the control cells.Conclusions:(1)During hippocampal development of rats,transcription of GAP-43 and Nogo-A gene was higher in embryos than other phases.GAP-43 mRNA expressed highly in senile rats experienced a lower stage after birth,while Nogo-A maintained in a high expression level in adult and senile rats.The results suggested that hippocampal neurons in senile rats still posses the powerful capability of growth and regeneration, and higher expression of growth inhibitors may be the capital barrier of nerve regeneration.(2)Transcription of netrin-1 gene,a growth and guidance factor,expressed highly from embryos to adults all the while except senile rats.Expression of Sema 3A mRNA was mainly confined to embryonic and neonate rats,however,Nrp-1,its receptor,was gradually increased after experienced a lower stage in neonate rats.(3)Rho-A and Rac-1,signaling molecules in cells,shared similar expressed pattern that demonstrated an evident phase expression with hippocampal development.The higher phase was in embryonic,juvenile and senile rats,otherwise,lower in neonate and adult rats.(4)Transcription of tubulin gene stepped down with development of hippocampus from embryos to adults,otherwise,stayed in the same level compared with adult rats. Although CRMP-1 gene possessed a similar pattern in transcription from embryos to adults,it expressed highly in senile rats compared with adult rats.(5)Activating Rho kinase could induce neurites to be collapsed and outgrowth to be retarded,and the results were the decreased number of neurites and shortened length. Contrast to activation,inhibition of Rho kinase induced neurites to branch and outgrow.The results suggested that Rho kinase not only participated in regulating neurite growth,but also regulating neurite branch formation by rearranging microtubules.Inhibiting activity of Rho kinase could reverse the function of LPA to retard neurite growth,otherwise,only inhibiting Rho kinase could not reverse the rearrangement of microtubules induced by LPA.(6)Finishing amplification of CRMP-1 cDNA and cloning vector of CRMP-1-PGEM-T Easy,and constructing a eukaryotic expression vector of CRMP-1-phrGFP Ⅱ-N.(7)Mter transfection of PC12 cells induced by nerve growth factor,CRMP-1 protein was detected in cell body and neurites.Expression of CRMP-1 protein induced cells to shorten neurites,suggesting CRMP-1 gene could inhibit neurites to outgrow and induce them to be collapsed.