Chin1 In Schwann Cells Controls Myelination By Regulating Actin Dynamics

The myelin sheath is a multilayered structure of lipids and proteins that wraps tightly around the axon.The myelin sheath is essential for accurate and rapid conduction of nerve impulses and provides essential metabolic support for axons.Failure to form or damage myelin sheaths can lead to a variety of neurological diseases.Oligodendrocytes form myelin in the central nervous system,while Schwann cells form myelin in the peripheral nervous system.When the Schwann cells form myelin sheaths,they stretch out the cytoplasmic membrane,making them thousands of times larger.The extension of the Schwann cells cytoplasmic membrane involves dramatic changes in the intracellular actin cytoskeleton.Surprisingly,however,little research has been done on the changes in the actin cytoskeleton and its regulatory mechanisms during the myelin sheath formation of Schwann cells.Actin cytoskeleton in eukaryotic cells is essential for normal function and survival of cells.Actin cytoskeleton plays an important role in a variety of cellular processes,such as motility and migration,cell division,vesicle transport,signaling,polarity formation,and epithelial maintenance of cellular barriers.For multicellular organisms,including in the process of embryonic development of cell morphology,the formation of the nervous system of the neurite formation and reshape the neurites,immune cell chemotaxis movement,fibroblast migration process such as after the injury,needs to be driven by actin cytoskeleton platy pseudopodia formation and depolymerization.Actin dynamics and reorganization are spatiotemporally regulated by a large number of actin binding proteins and upstream signaling molecules.Actin filaments and actin binding proteins cooperatively construct organized cytoskeletal structures in cells,with different shapes,stabilities and functions.Cofilin1 is a key regulator of actin filament dynamics,and it stimulates depolymerization and severing of actin filaments.In vivo,cofilin1 is inactivated by phosphorylation of Ser-3 by LIM kinase(LIMK)and testicular protein kinase(TESK),The phosphorylated cofilin1(pcofilin1)is dephosphorylated and reactivated by the Slingshot(SSH)family of protein phosphatasesOur lab found a new gene named Chin1 in the process of inducing epileptic cell model by DNA microarray.Our lab unpublished study found Chin1 can regulate myelination in the central nervous system(CNS),but it is unknown whether Chin1 regulates myelination in the peripheral nervous system(PNS).In order to study the function of the new gene Chin1,we first constructed Chin1 knockout HEK293T cells by using CRISPR/Cas9 technology,and verified the Chin1 is successfully knockout by gene sequencing and western blot.In the process of culturing knockout cells,we unexpectedly found that the adherent ability of knockout cells was weakened.By referring to relevant papers,we speculated that Chin1 might participate in the remodeling process of actin cytoskeleton.In order to verify the above conjecture,F-actin in Chin1 knockout cells was detected by rhodamine-phalloidin dye,and we found that F-actin significantly increased after Chin1 knockout.This result was also verified by a G-actin/F-actin separation experiment.At the same time,in order to study the role of Chin1 in the myelination process of Schwann cells,we used the Cre-Lox P system to construct transgenic mice with Chin1 specifically knocked out in Schwann cells.We also found F-actin significantly increased in knocked out mice by using rhodaminephalloidin dye.These findings suggest that Chin1 is involved in the regulation of actin cytoskeleton remodeling.Then,we found the F-actin could polymerization normally after Chin1 knockout,it means Chin1 regulates F-actin depolymerization.Cofilin1 is a key regulator protein in the depolymerization of F-actin.We found the p-cofilin1 is increased in Chin1 knockout mice and HEK293 T cells by immunofluorescent staining and western blot.This results demonstrate that Chin1 can regulate the phosphorylation of cofilin1.According to our lab’s unpublished results that Chin1 knockout can reduce the activity of mTORC1,but there is no direct studies have reported whether the mTORC1 signaling pathway has a regulatory effect on cofilin1 or actin dynamics.In order to answer whether Chin1 regulates cofilin1 or actin dynamics through the mTORC1 signaling pathway,we performed the following tests: 1.inhibiting the mTORC1 activity by treating with Rapamycin in cultured HEK293 T cells.Immunofluorescence staining and western blot analysis showed that the activity of mTORC1 is decreased,but Chin1 and p-cofilin1 were not alter after treating with Rapamycin,so was the F-actin.2.Rescue the mTORC1 activity by overexpressing myc-Rheb1(S16H)in Chin1 knockout HEK293 T cells,western blot shows the mTORC1 activity is rescued,but p-cofilin1 and F-actin were not.3.Establishing Schwann cell specific Chin1 knockout and myc-Rheb1(S16H)knockin transgenic mice.Immunofluorescence staining and western blot show the mTORC1 activity is rescued,but p-cofilin1 and F-actin were not.This results demonstrate that Chin1 regulate the phosphorylation of cofilin1 independent of mTORC1.The decrease of mTORC1 activity is usually accompanied by the increase of mTORC2 activity feedback.However,the mTORC2 signaling pathway can regulate the cytoskeleton.The mTORC1 activity is decreased after Chin1 knockout,so we detected the mTORC2 activity.Immunofluorescence staining and western blot show the mTORC1 activity is decreased,but the mTORC2 activity is not increased.The mTORC2 regulates cytoskeleton by phosphorylating PKC family protein,LIMK1 regulating by PKC can phosphorylate cofilin1,we found p-LIMK1 was not alter in Chin1 knockout mice and cell by western blot.This results demonstrate that Chin1 regulate the phosphorylation of cofilin1 and F-actin dynamics independent of mTORC2.How does the Chin1 regulate the phosphorylation of cofilin1? We found pLIMK1 and p-SSH1 were not alter,witch were phosphorylating and dephosphorylating cofilin1 respectively.Therefore,we speculate that Chin1 may interact directly with cofilin1 to regulate LIMK1 phosphorylation.GST pull-down detecting shows Chin1 can interact directly with cofilin1.By PLA assay we found Chin1 can interact directly with cofilin1 in situ.Finally,through in vitro kinase experiments,we found that the combination of Chin1 and cofilin1 inhibited the phosphorylation of cofilin1 by LIMK1.This results demonstrate that Chin1 inhibits LIMK1 phosphorylation of cofilin1 by direct interaction with cofilin1.The most important function of Schwann cells is to wrap the axons and form myelin sheath.If glia cells fail to depolymerized actin normally,the inner structure of the myelin sheath cannot be eliminated,and eventually the myelin sheath cannot form normally.We detected the myelin structure of four-week-old mice by using EM,and we found the inner tongue could not disappear normally in Chin1 knockout mice,accompanied by the thinning of myelin sheath;by calculating the g-ratio value of nine-month-old mice,it was found that the myelin was thin in Chin1 knockout mice.This results demonstrate that the specific knockout of Chin1 in Schwann cells prevented the inner tongue structure from disappearing,resulting in permanent myelin defects.Myelin defects in mice often show behavioral abnormalities such as limb gait instability and shortened step length.Therefore,we finally tested the behavioral characteristics of Chin1 knockout mice in Schwann cells.The Digi Gait analysis system showed that the suspension time of the hind limbs increased,while the landing time decreased.Step length decreases step frequency increases;at the same time,the hind legs of the mice were enlarged.This results demonstrate that the myelin deficiency caused by Chin1 knockout in Schwann cells has influenced the behavior of mice.In this study,we will construct Chin1 knock out HEK293 cells and transgenic mice with Chin1 knocked out specifically by Schwann cells,and conduct biochemical tests on them to clarify the role of Chin1 in the regulation of cytoskeleton and the influence of Chin1 on the myelin sheath formation of Schwann cells.The molecular mechanism by which Chin1 regulates the phosphorylation of cofilin1 was elucidated through in vitro GST pull-down experiment and in vitro kinase experiment.Conclusions1.Chin1 affects actin cytoskeletal remodeling by regulating the phosphorylation of cofilin1.2.Chin1 regulates the phosphorylation of cofilin1 independent of mTORC1/mTORC2.3.Chin1 inhibits LIMK1 phosphorylation of cofilin1 by direct interaction with cofilin1.4.Ablation of Chin1 in Schwann cells prevented the depolymerization of f-actin in the inner tongue,resulting in permanent myelin defects.5.Ablation of Chin1 in Schwann cells resulted in abnormal behavior in the mice.Innovation and significance The remodeling of actin cytoskeleton is very important for cells to perform their biological functions.In Schwann cells,failure to depolymerize actin bundles normally will result in permanent myelin defects and affect normal behavior.This study found that the new gene Chin1 is essential for the remodeling of actin cytoskeleton.Chin1 affects the remodeling of actin cytoskeleton by regulating the phosphorylation of cofilin1,and elucidated the regulation mode of Chin1 protein on the phosphorylation of cofilin1.This study will contribute to the in-depth understanding of the effects of intracellular related molecules on actin cytoskeleton remodeling,and provide new theoretical basis and potential therapeutic targets for relevant drug research.