| Spinal cord injury(SCI)is a serious central nervous system disease causing paralysis and sensorimotor impairments,which affects the function of multiple systems such as cardiovascular,digestive,urination,etc.Severe SCI can lead to completely loss of feeling and motor function below the injured level.SCI not only bring about great physical and mental pain to patients and their families,but also lots of labor loss to the community.Thus,it becomes urgent need to overcome sophisticated medical problems,and to find effective treatment of SCI.The pathogenesis of SCI is complicated which consists of primary and secondary injury mechanisms and has not been clearly stated yet.The primary injury occurs simultaneously at the accident,leading to neuron death directly.While the secondary injury is characterized by extensive and persistent pathological stress,including ischemia,oxidative stress,ionic homeostasis disturbances and inflammatory responses.Finally,the overloaded pathological stress results in worse neuron viability around the primary lesion site,further locomotor dysfunction and retarded post-traumatic recovery.Though we can't avoid the accident if it has happened and the primary injury afterwards,but what we can do is to save more neurons during secondary injury.Based on the context,although the exact molecular pathway of the secondary injury still remains elusive,removal of these deleterious stress response factors during secondary injury might promote post-traumatic spinal neuron survival.Therefore,it is significant to investigate the pathological response and mechanism of secondary spinal cord injury mechanism.Arguably,one of the most intriguing and compelling aspects of contemporary biology is the concept of autophagy,which translates from the Greek words auto(self)and phagein(to eat),meaning self-eating.Autophagy is an ancient catabolic process by which cells recycle macromolecular proteins and damaged organelles(such as mitochondria)by an elaborate autophagolysosomal pathway.Autophagy is rapidly induced during nutrient deprivation and ischemia,presumably as an initial survival mechanism to subvert the accumulation of lethal protein aggregates and damaged organelles.Autophagy may be involved in the secondary injury of SCI,therefore,recently,more and more studies have focused on the mechanism of autophagy after SCI.The mammalian Ste20-like kinase 1(Mst-1)is a serine-threonine kinase and a component of Hippo tumor suppressor pathway,which reacts to pathologically relevant stress and regulates cell death.In previous study,Lee et al showed that activated Mst-1 upregulated in the motor neurons of amyotrophic lateral sclerosis(ALS),and genetic deficiency of Mst-1 not only delayed symptom onset and mortality,but also improved the viability of spinal motor neurons,implicating that Mst-1 may function as a key modulator of neurodegeneration.Recently,Maejima et al.showed that Mst-1 inhibits autophagy and promotes cell death after myocardial infarction,suggesting a key determinant for Mst-1 in inhibiting autophagy flux and attenuating cell survival during detrimental stress response.However,little is known about its role in SCI.Inspired by these findings,we carried out study to investigate whether Mst-1 deficiency could enhance autophagy flux,through which injured spinal motor neurons adequate degraded toxic protein aggregates,and eventually,lead to more motor neuron survival.Part 1:Mst-1 deficiency promotes spinal motor neuron survival after SCIObjective:The mammalian Ste20-like kinase 1(Mst-1)is a serine-threonine kinase and an important upstream molecule of Hippo tumor suppressor pathway,which reacts to pathologically relevant stress,autophagy and regulates cell death.Some studies have shown that Mst-1 deficiency promotes injured spinal motor neuron and cardiocyte survival through enhancement of autophagy flux.However,little is known about its role in SCI.Therefore,we carried out study to investigate the effect of Mst-1 deficiency on mice model of SCI.Methods:1.Firstly,all mice were subjected to spinal T9 complete transection.2.Western blot analysis of Mst-1 and p-Mst-1 expression in the sham-operated and 4 weeks post-injury wild type groups.3.Co-immunostaining of Mst-1 and p-Mst-1 with ChAT on coronal sections in the sham-operated and 4 weeks post-injury wild type groups.To investigate the expression of Mst-1 and p-Mst-1 in ChAT positive motor neurons.4.Co-immunostaining of ChAT and GFAP ranging from rostral 600 ?m to caudal 600 ?m around the epicenter.To detect the number of ChAT positive motor neurons in Mst-1+/+ and Mst-1-/-groups 4 weeks post-injury.5.FG was injected into the gastrocnemius and retrogradely label spinal motor neurons ranging from rostral 600 ?m to caudal 600 ?m around the epicenter in Mst-1+/+and Mst-1-/-groups 4 weeks post-injury.6.Timeline of the change in BMS scores in Mst-1+/+ and Mst-1-/-mice to investigate the locomotion recovery of mice after SCI.7.Co-immunostaining of vGlutl and ChAT showing synapse reformation between the glutamatergic terminals and motor neurons in Mst-1+/+ and Mst-1-/-mice 9 weeks post-injury.Results:1.We successfully established a spinal T9 complete transection model in mice.2.No significant differences in total Mst-1 expression were detected in the injured group compared with the intact group.While,the phosphorylated Mst-1 was strongly upregulated in the lesion area of the injured group compared with the intact group.3.No significant differences in total Mst-1 expression were detected in the spinal motor neurons of the injured group compared with the intact group.While,the phosphorylated Mst-1 was strongly upregulated and was specifically activated in motor neurons after SCI.4.Homozygous deletion of Mst-1 increased the amounts of survived spinal motor neurons after SCI,which implicated that Mst-1 deficiency promoted post-traumatic spinal motor neuron survival.5.FG tracing data showed that more FG positive neurons were detected in Mst-1-/-mice than in Mst-1+/+ mice.These results not only demonstrated Mst-1 deficiency promoted post-traumatic spinal motor neuron survival,but also indicated that these motor neurons have intact connection with its innervated effector muscles which might lead to better locomotor function.6.In both Mst-1+/+ and Mst-1-/-mice,hind limb locomotion was severely impaired for the first 7 days post-injury,then slightly improved over time,and reached a plateau 56 days later.Notably,Mst-1-/-mice showed higher BMS scores than Mst-1+/+ mice at the time point of 49,56 and 63 days post-injury.7.Glutamatergic interneurons formed more glutamatergic synapses in contact with ChAT positive motor neurons in Mst-1-/-mice compared with Mst-1+/+ mice.Conclusion:Here,we found that p-Mst-1,the activation form of Mst-1,was induced in the post-traumatic spinal motor neurons,and Mst-1 deficiency promoted the survival of post-traumatic spinal motor neuron,BMS scores,and synapse reformation.Part 2:The mechanism of Mst-1 deficiency promotes spinal motor neuron survival after SCIObjective:Some studies demonstrated that Mst-1 deficiency induces autophagy and promotes cardiomyocytes survival in myocardial infarction.Based on this report,we assumed whether Mst-1 deficiency plays a role in promoting the survival of post-traumatic spinal motor neuron through the enhancement of autophagy flux.Therefore,we tested the expression of some autophagy related proteins,and investigate the state of autophagy flux to figure out the mechanism of Mst-1 deficiency promotes spinal motor neuron survival after SCI.Methods:1.Western blot analysis of LC3-? expression pattern in the Mst-1+/+ and Mst-1-/-lesion sites from 1 day to 4 weeks post-injury.2.We choose the highest LC3-? expression on 7 days post injury,western blot analysis of LC3-II and p62 expression in the sham-operated and 7 days post-injury lesion area of the Mst-1+/+ and Mst-1-/-mice were performed to test the state of autophagy flux.3.Immunostaining of LC3-? and p62 expression in ChAT positive motor neurons in the sham-operated and 7 days post-injury lesion area of the Mst-1+/+and Mst-1-/-mice.To test the state of autophagy flux in ChAT positive motor neurons.4.The number of ChAT positive motor neurons in the Mst-1+/+ and Mst-1-/-mice 4 weeks post-injury after Baf treatment were counted.To verify autophagy flux was essential for the survival of injured spinal motor neurons in Mst-1 deficiency mice.5.Western blot analysis of LC3-? and p62 expression in the lesion area of the Mst-1+/+ and Mst-1-/-mice 7 days post-injury.To verify the state of autophagy flux after Baf treatment.6.Immunostaining of LC3-II and p62 expression in ChAT positive motor neurons in the lesion area of the Mst-1+/+ and Mst-1-/-mice 7 days post-injury.To verify the state of autophagy flux after Baf treatment.Results:1.Upregulation of LC3-? was detected in both Mst-1+/+ and Mst-1-/-groups from 1 day to 4 weeks post-injury,and this upregulation reached a peak on the 7 days post-injury and gradually decreased until at least 4 weeks post-injury.while the Mst-1-/-group showed higher LC3-? expression than Mst-1+/+ group at the same time points.2.At 7 days post-injury when the expression of LC3-? reached a peak,Mst-1-/-mice showed more LC3-? formation and less p62 accumulation than Mst-1+/+ mice did,indicating an enhanced autophagy flux in Mst-1-/-mice.3.Our results provided more LC3-? formation and less p62 accumulation in Mst-1-/-spinal motor neurons than in Mst-1+/+ spinal motor neurons,indicating an enhanced autophagosome formation and autolysosome degradation in spinal motor neurons of Mst-1-/-mice,which were consistent with the western blot results above.4.Immunostaining of ChAT revealed that the number of survived spinal motor neurons was decreased in both Mst-1+/+ and Mst-1-/-groups with Baf treatment.However,the number of survived spinal motor neurons in Mst-1+/+ group was less than it in Mst-1-/-group after Baf treatment.These data revealed that the number of survived motor neurons was decreased with Baf treatment after SCI,and Mst-1 deficiency could minimize the neuron loss with Baf treatment.5.The increased level of p62 and LC3-? were detected in Baf group,indicating Baf succeeded in suppressing the autolysosome degradation of autophagy-lysosome pathway.In addition,the Mst-1-/-group showed higher autolysosome degradation than in Mst-1+/+ group,suggesting that Mst-1 deficiency could correct the suppression of autolysosome degradation with Baf treatment in a certain range.6.The increased level of p62 and LC3-? in spinal motor neurons were detected in Baf group,indicating Baf succeeded in suppressing the autolysosome degradation of autophagy-lysosome pathway.In addition,the Mst-1-/-group showed higher autolysosome degradation than in Mst-1+/+ group,suggesting that Mst-1 deficiency could correct the suppression of autolysosome degradation with Baf treatment in a certain range.Conclusion:Autophagy was induced after SCI,and autophagy flux was disrupted after SCI,while Mst-1 deficiency could correct this disruption in a certain range via more autophagosome formation and enhancement of autolysosome degradation.Through which injured spinal motor neurons adequate degraded toxic protein aggregates,and eventually,lead to more motor neuron survival.Taken together,our findings demonstrate that autophagy flux is essential for Mst-1 deficiency to promote the survival of spinal motor neurons after SCI. |
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