Role And Mechanism Of TLR4 Signaling Pathway On Neurogenesis Following Traumatic Brain Injury

As one of the leading life-threatening diseases,TBI often causes high mortality and severe neurological deficits due to the inadequate self-repair capacity of CNS.Therefore,TBI is a common public health challenge for all humankind.With the improvement of TBI therapy in recent years,the survival time of TBI patients has increased significantly,there are still a large number of patients who have been suffering from neurological disability or cognitive impairment in their whole life.TBI creates a heavy burden on patient individuals,their families and the society.Therefore,it is important to investigate neurogenesis and seek reasonable intervention strategies for the improvement of TBI outcomes.Studies have shown that endogenous neurogenesis in hippocampus occurs continuously throughout life.As the core cell of brain reconstruction following trauma,the NSCs in SGZ of hippocampus can be activated after TBI,and then restore the damaged neural function through proliferation,differentiation,migration,integration and synaptic plasticity.However,endogenous neurogenesis of NSCs is not sufficient to repair the injury caused by trauma due to the poor pathological environment in the injuried brain.Therefore,strategies are required to activate effectively endogenous NSCs to generate more functional neurons after TBI for brain rebuilding and functional restoration.Many factors and mechanisms contribute to manage the potential of NSCs in neurogenesis process.It is well known that neuroinflammation,one of the prominent pathological responses in injured brain,is a double edged sword for neurogenesis depending on the balance between neurotoxic and neuroprotective effect in hippocampus.On the one hand,inflammation is propitious to initiate NSC proliferation and differentiation in pathological status of CNS,on the other hand,inflammatory cytokines in neurogenic niche are detrimental to NSC survival and fate.Therefore,understanding the key molecules and its signaling pathway of neuroinflammation is of great significance to ameliorate neural regeneration following TBI.As an important member of pattern-recognition receptor family,TLR4 has been found in diverse cell types including microglia,astrocyte,and neuron in CNS.A growing body of evidence suggested that TLR4 signaling pathway played a key role in the inflammation of CNS diseases.Therefore,it has been proposed as a therapeutic target.TLR4 recognized not only pathogen-associated molecular patterns(PAMPs)but also damage-associated molecular patterns(DAMPs),and then induced intracellular cascade activation and release inflammatory cytokines in response to the pathological condition of injured brain.In early years,researches on TLR4 signal pathway in CNS focused mainly on its effect on microglia and neuron.Recently,several groups have provided evidence to support the crucial role of TLR4 signaling pathway in governing NSC proliferation and differentiation.However,the underlying mechanism between TLR4 signaling and NSCs potential is still unknown.Moreover,the involvement of TLR4 signaling pathway on endogenous NSCs biological characteristics in hippocampus after TBI has not been reported.Therefore,the current study is intended to investigate the effect of TLR4 on hippocampal neurogenesis in experimental TBI mice model.In addition,the mechanisms of TLR4 downstream cascade in NSCs proliferation and differentiation were also explored.The new perspective of understanding endogenous NSCs will be helpful for novel strategies to improve brain rehabilitation and functional recovery after TBI.ObjectiveThe first part of this study was aimed at exploring TLR4 expression and NSCs proliferation in hippocampus after TBI in vivo.The second part was designed to analyze the effect of TLR4 expression alteration on NSCs proliferation and differentiation post trauma in vivo and in vitro.The third part was aimed to elucidate the putative mechanism of TLR4 pathway in NSCs neurogenesis for brain restoration in vivo and in vitro.MethodsOn the basis of TBI mice model establishment in vivo and NSCs culture in vitro,immunofluorescence staining,Western blotting and RT-PCR were carried out to detect the expression of TLR4 and NSCs proliferation in hippocampus after TBI.Then,TLR4 KO mice and TLR4 pharmacological antagonist/agonist were used to modulate TLR4 expression in vivo and in vitro.The effect of such an expression change on NSCs survival,proliferation and differentiation after TBI was investigated by immunofluorescence staining,CCK-8 detection,Western blotting,transmission electron microscopy and laser confocal microscope observation.Furthermore,specific pharmacological inhibitors and siRNA interfering technology were employed to inhibit Myd88 and TRAM in the downstream of TLR4 in vivo and in vitro.FCM and immunofluorescence staining were taken to observe the proliferation and differentiation of NSCs.Morris water maze test was used to evaluate neurological function post trauma.The level of TLR4 downstream molecules and inflammatory cytokines were,respectively,detected by Western blotting and ELISA.Results 1.Compared with sham group,the number of hippocampal BrdU/TLR4 positive cells in TBI group increased evidently.Quantitative assessment showed that the population of BrdU/TLR4 positive cells increased at 3 day,maximized at 7 day,and then decreased to 28 day post trauma.Expression of hippocampal TLR4 protein was up-regulated at 3 day post trauma and reached the peak at 7 day,then descended gradually over 14,21 and 28 day,and still maintained at a higher level than the sham.TLR4 mRNA expression also increased at 3 day post trauma and rose to the peak at 7 day,and fell gradually over the rest of the survival periods to a level still higher than the sham.Statistical analysis showed that there was a correlation between NSCs proliferation and TLR expression after TBI.2.Compared with WT mice,the number of hippocampal BrdU/SOX2 positive NSCs in TLR4 KO mice increased significantly after TBI.Similarly,the number of BrdU/DCX and BrdU/NeuN positive neurons also increased while BrdU/GFAP positive glial cells decreased in TLR4 KO mice.In addition,NSCs and neurons respectively labeled by Nestin and TUJ1 in TAK-242 group were higher than that of control group,but the number of positive glia cells stained by GFAP and O4 decreased in vitro.LPS treatment induced lower NSCs neurospheres formation,proliferation,neuronal differentiation,but higher glial differentiation compared to control group in vitro.3.Compared with TBI group,BrdU/SOX2 and BrdU/NeuN positive cells increased dramatically in TBI+ST group.BrdU/SOX2 positive cells also increased in TBI+RES group,but BrdU/NeuN neurons maintained at the same level with TBI group.Learning and memory ability enhanced in TBI+ST mice,but no changes were observed in TBI+RES mice.Additionally,Myd88 siRNA administration resulted in an increase of NSCs proliferation and neuronal differentiation,a decrease of glial differentiation,as well as reduction of anti-inflammatory and pro-inflammatory cytokines in NSCs culture medium compared to LPS group in vitro.TRAM siRNA treatment acted only on NSCs proliferation and anti-inflammatory cytokines but not NSCs differentiation or pro-inflammatory cytokines in vitro.ConclusionIt was found that TLR4 expressed on NSCs in SGZ of hippocampus in vivo,and TLR4 expression was correlated with NSCs proliferation in the process of endogenous neurogenesis after TBI.Inhibition of TLR4 signaling led to an increase of NSCs proliferation and differentiation into neuron,which promoted the neural regeneration after TBI.TLR4-Myd88 pathway was involved in governing both proliferation and differentiation of NSCs,and TLR4-TRIF pathway only worked on NSCs proliferation but not on differentiation fate,which can be ascribed to the diverse level of anti-and pro-inflammatory cytokines in the downstream of two pathways.