Study On The Function Of STAT3 And SOCS3 In Neurite Growth

After injury to the adult central nervous system (CNS), injured axons usually fail to regenerate, whereas following injury to the mature peripheral nerve system (PNS), damaged axons regenerate successfully if not to appropriate targets. Two major obstacles have been identified to account for this poor regeneration in the mature CNS. One is an unfavourable glial environment for axonal growth, characterised by the presence of myelin debris, glial scar, cavitation and lack of facilitatory molecules. The other is the failure of damaged CNS neurons to initiate a programme of gene expression which is beneficial for axon regeneration. Accumulating evidence indicates that overcoming of inhibitory signals is beneficial but must be combined with activation of the intrinsic growth state of neurons to achieve successful axon regeneration.A favourable neuronal population in which to compare regeneration in the CNS and PNS is the collection of the primary sensory neurons of the dorsal root ganglia (DRGs). These neurons have both central and peripheral processes. If the peripheral branch is damaged, robust regeneration is observed and results in functional recovery. In contrast, axons in the central branch regenerate poorly after lesion. In the 1970s, it was shown that axonal regeneration in the sciatic nerve of adult mice or rats is enhanced if the nerve is crushed or transected 2-28 days before a subsequent more proximal sciatic nerve injury. In the 1980s, it was further demonstrated that regeneration of central axons in the dorsal roots can be accelerated 3-fold by a previous conditioning peripheral nerve lesion and that the number of central axons growing into a peripheral nerve graft after a dorsal column transection can be increased 100 times by a concomitant peripheral nerve lesion. Moreover a peripheral nerve lesion 1-2 weeks earlier results in regeneration of central branches into and beyond the spinal dorsal column lesion site.Furthermore, it was proved that previous in vivo peripheral axotomy activated the subsequent in vitro growth capacity of DRG neurons. Therefore the growth enhancing effect must be the result of a neuronal response induced by the conditioning lesion, such as activation of transcription in the nerve cell body.Signal transduction is the current hot spot of axonal regeneration after nerve injury. Signal transducer and activator of transcription 3 ( STAT3) is activated by various cytokines and growth factors and involved in multiple physiological processes.STAT3 proteins have conservative domains including DNA binding2domain , SH2 domain and transcription activation domain. STAT3 proteins are activated by tyrosine phosphorylation at Tyr705 ,then form dimmers , translocate to the nucleus , and bind to specific DNA2response element of target genes to induce gene transcription. STAT3 is expressed during the development of nervous system , and regulates the differentiation of neural progenitor cells with synergistical contribution of other signaling cascades. The methylation status of STAT3 binding element also determines cell lineage specification.STAT3 protein also plays important roles in maintaining nerve cells survival and promoting their regeneration after injury. It has been shown that JAK/STAT signalling is activated after peripheral axotomy and is critical for neuronal regeneration and survival of neurons. After peripheral nerve transection, STAT3 protein in regenerating motoneurons is significantly increased and activated by Tyr705 phosphorylation. STAT3 phosphorylation after sciatic nerve transection can be blocked by sustained perineurial infusion of the JAK2 inhibitor AG490 to the proximal nerve stump. Furthermore, perineurial infusion of AG490 compromised neurite outgrowth in vitro and significantly attenuates dorsal column axonal regeneration in the adult spinal cord after a conditioning sciatic nerve transection. These results support the hypothesis that STAT3 activation is necessary for increased growth ability of DRG sensory neurons after a conditioning injury.SOCS3 is a suppressor of JAK/ STAT signal transduction pathway and indirectly regulates STAT3 phosphorylation , as one in the family of suppressor of cytokine signalling (SOCS) comprises 8 members: CIS (cytokine inducible SH2 protein) and SOCS1-SOCS7. SOCS3 can bind to gp130 through the phospho-tyrosine motif, and block gp130 signalling possibly through interfering with its tyrosine phosphorylation of gp130 to inhibit STAT-mediated gene expression. Measurements of SOCS3 mRNA in rat dorsal root ganglia showed a significant induction in this inhibitory molecule after peripheral nerve injury.The aim of this projectis is to construct lentiviral vector carrying STAT3 and mutated mouse oestrogen receptorα(ER), and infect neurons of rat dorsal root ganglia to investigate functions of STAT3 in adult rat primary sensory neurons. Also the functions of SOCS3 in adult rat primary sensory neurons were investigated in vitro through transduction of lentiviruses yielding a native SOCS3, or a mutant SOCS3 with dominant- negative actions.In this study,gene recombinant technology was employed to cleave and insert the PmeI fragment of STAT3ER-IRES-GFP into the PmeI site of the lentivirus transfer vector pRRL-MCS+ , to construct a lentiviral vector pRRL-STAT3ER-IRES-GFP.RT-PCR,western blotting and sequencing analysis were used for identification. Then, 293T cells were transfected with pRRL-SOCS3-IRES-GF , pRRL-mSOCS3-IRES-GFP and pRRL- STAT3ER-IRES-GFP separately, and the titer was determined. The peripheral and dorsal roots of rat left L5 DRG were cut near their ganglia,L 5 DRGs were taken to separate neurons which were cultured in vitro. For positive control, the left sciatic nerve (SN) was transected at the hip immediately before dorsal root crush.SOCS3 mRNA within DRGs neurons were detected by real time PCR and In situ hybridisation . Neurons infected by lentiviruses were observed by fluorescentmicroscopy to detect the ratio of nuclear to cytoplasmic phosphorylated STAT3 immunoreactivity and neurite lengths.Results as follow: The lentiviral vector plasmid pRRL-STAT3ER- IRES-GFP was identified correct by RT-PCR and western blotting, and DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by Genbank. After infection, the concentration of the virus titer was 1010-11 TU /mL. After infection with pRRL-STAT3ER- IRES-GFP, nuclear translocation of STAT3ER in infected neurons was seen obviously after stimulation with 4HT. Neurite outgrowth was enhanced by STAT3. The concentration of SOCS3 mRNA within DRG neurons increases considerably after nerve injury, SOCS3 construct was effective in blocking nuclear accumulation of endogenous STAT3 and mutant SOCS3 enhanced neurite growth.Conclusion Lentiviral vector carrying STATSER was successfully constructed and activation of STAT3 signaling is beneficial to axonal growth .SOCS3 is detrimental to axonal growth through inhibition of STAT3 and/or other transcription factors.