The Role And Mechanism Of SIRT6 In Peripheral Nerve Schwann Cell Differentiation And Dedifferentiation

Schwann cells are the unique glia cells that surround axon and form myelin with a lamellar structure,myelin play an important role in protecting axon,conducting action potential,and transporting substances.Abnormal myelin development can cause sensory and motor dysfunction.Schwann cells have a high plasticity.During the myelin development,it undergoing proliferation,migration and differentiation.After peripheral nerve injury,Wallerian degeneration will occurs in the denervated distal nerve,accompanied by axon and myelin degeneration,and Schwann cells convert into a dedifferentiated phenotype,also known as "repair" cell,which assist the macrophages to remove the tissue debris,and regain the ability to proliferate and re-differentiation.Based on this property,peripheral nerve exhibit stronger regenerative capacity than central nerve.There are various transcriptional factors involved in Schwann cell differentiation and dedifferentiation.For instance,Schwann cell differentiation requires the expression of Krox20 and Sox10;Schwann cell dedifferentiation requires the expression of c-Jun,Sox2 and NF-κB.SIRT6 is a nicotinamide adenine dinucleotide（NAD+）-dependent deacetylase,which plays an important role in anti-aging,promoting DNA repair,maintaining energy metabolism,inhibiting inflammation,and regulating lipid metabolism.The development of peripheral myelin is premised on Schwann cell differentiation that precisely regulated by transcriptional factors and cholesterol-rich membrane synthesis.Previous studies have shown that SIRT6,as a co-transcriptional factor,directly or indirectly regulates the expression or activation of transcriptional factors.In addition,SIRT6 regulates cholesterol synthesis.Therefore,we speculate that SIRT6 may affect Schwann cell differentiation and dedifferentiation by steering cholesterol synthesis or regulating the expression of transcriptional factors.Currently,there is no literature on the expression pattern and role of SIRT6 in the peripheral nerve.We put forward some scientific questions:is SIRT6 expressed in the peripheral nerves and what is SIRT6 expression pattern during myelin development and after peripheral nerve injury?And what role does SIRT6 play in Schwann cell differentiation during myelin development and dedifferentiation after nerve injury,and what are the underlying mechanisms?Objective:To explore the role and mechanism of SIRT6 in Schwann cell differentiation and dedifferentiation,and provide a new theoretical basis for myelin development and nerve regeneration in the peripheral nerve.Methods:1.The sciatic nerves of C57BL/6 postnatal mice at different ages were isolated,and the spatiotemporal expression pattern of SIRT6 in the developing sciatic nerve were detected by Western blotting and immunofluorescence.Primary Schwann cells were obtained from spinal nerves of C57BL/6 mice,dbcAMP and heregulin 1βwere added to induce cells to obtain a differentiated phenotype.Western blotting and immunofluorescence were used to detect the SIRT6 expression after Schwann cells differentiation.At the tissue and cellular levels,to investigate the SIRT6 expression in the differentiated Schwann cell.2.shRNA lentivirus targeting SIRT6 mRNA was constructed,and micro-injected into the sciatic nerve of C57BL/6 postnatal mice,to silence the SIRT6 expression in Schwann cells.Western blotting and immunofluorescence were used to detect the expression of SIRT6,MAG and MBP,and the ultrastructure of myelin was observed by transmission electron microscope,to explore the role of SIRT6 in myelin development;In vitro,Schwann cells were transfected with shRNA lentivirus or treated with SIRT6 inhibitor OSS₁28167,to induce differentiated phenotype subsequently,then the expression levels of Schwann cell immature markers（p75 and c-Jun）and mature markers（Krox20 and MAG）were detected,to explore the influence of SIRT6 silencing on Schwann cell differentiation.3.To identify whether SIRT6 affects Schwann cell differentiation and myelination is via cholesterol synthesis.And squalene synthase（SQS or FDFT1）,an important rate-limiting enzyme for cholesterol synthesis was detected,to explore the effect of SIRT6 silencing on FDFT1 expression and cellular cholesterol synthesis.4.qPCR and Western blotting were used to detect the expression changes of SIRT6 at different time points after sciatic nerve injury.Using sciatic nerve explants,an in vitro Wallerian degeneration model,SIRT6 inhibitor OSS₁28167,SIRT6 activator UBCS039 and JNK inhibitor SP600125 were added to this model.The expressions of NF,MBP,MAG and c-Jun were examined by Western blotting and immunofluorescence of tissue sections or Teased fiber,to explore the effect of SIRT6 activity changes on Wallerian degeneration;Using an in vitro Schwann cell dedifferentiation model,OSS₁28167 was added to explore the effect of SIRT6 on Schwann cell dedifferentiation;Immunoprecipitation was used to identify the interaction of SIRT6 and c-Jun,and to explore whether SIRT6 targets c-Jun to regulate Wallerian degeneration and Schwann cell dedifferentiation after injury.Results:1.SIRT6 expression was comparatively low in P4 sciatic nerve,its expression in P7P14 sciatic nerve was gradually increased and reached a peak at P14,and SIRT6 expression gradually decreased from P21-8 W,and maintained a low level in mature sciatic nerve.SIRT6 was specifically expressed in the myelin but absence in axon.SIRT6 expression was significantly increased in differentiated Schwann cells in vitro.These data suggest that SIRT6 may involve in Schwann cell differentiation and myelination.2.shSIRT6 lentivirus micro-injection significantly reduces SIRT6 expression in Schwann cells in vivo,results in a thinner myelin and reduces MAG and MBP expression.And SIRT6 inhibition by shSIRT6 transfection or OSS₁28167 treatment depress Schwann cell differentiation.These data suggest SIRT6 inhibition impairs Schwann cell differentiation and myelination.3.Protein-Protein interaction by STRING website found that SIRT6 may involve in Schwann cells cholesterol synthesis.SIRT6 silencing inhibits the expression of cholesterol synthesis pivotal genes including Hmgcr,Fdft1,Idi1,and Lss,and decreases free cholesterol level in Schwann cells.4.SIRT6 expression after peripheral nerve injury was gradually increased from 3 dpi to 7 dpi,reached the peak at 7 dpi,and then gradually decreased to a low level.Inhibition of SIRT6 activity accelerates Wallerian degeneration process（including axonal,myelin degeneration,and Schwann cell dedifferentiation）;Conversely,increasing SIRT6 activity delays the above process.5.SIRT6 directly targets c-Jun,the key transcriptional factor of Schwann cell dedifferentiation,and SIRT6 silencing increases c-Jun expression,which negatively regulates Wallerian degeneration.Conclusions:1.SIRT6 expression is increased after Schwann cell differentiation and during the critical period of myelin development.SIRT6 silencing inhibits Schwann cell differentiation and leads to hypomyelination,which is due to SIRT6 inhibition reduces cholesterol synthesis in Schwann cell.2.SIRT6 directly targets the transcriptional factor c-Jun to negatively regulate the process of Wallerian degeneration after peripheral nerve injury,including axonal and myelin degeneration and Schwann cell dedifferentiation.