TRIM31 Exacerbates Cerebral Ischemic Injury By Inhibiting Autophagy Through TLR9 Degradation

Stroke is the second leading cause of death and the dominant reason of disability worldwide.Stroke can be broadly divided into ischemic and hemorrhagic stroke.Ischemic stroke accounts for most cases of it.At present,clinical treatment strategies for acute ischemic stroke are thrombolytic therapy and thrombectomy,which are limited by their narrow therapeutic window.Therefore,it is pivotal to deeply explore the physiopathological mechanism of cerebral ischemia.Autophagy is known as programmed cell death.Moderate autophagy can clear damaged organelles,and protect cells against various injuries.However,long-term excessive autophagy brings redundant degradation of cell contents,contributing to cell death and eventually serious damage to tissues and organs.There are increasing evidences indicate that autophagy may be a new target in the treatment of ischemic stroke.TRIM31(Tripartite motif-containing protein 31)belongs to the E3 ubiquitin ligase TRIM family.It can specifically modify and degrade target proteins and participate in a variety of cell biological processes.The role of the TRIM31 in cerebral ischemia has rarely been reported.Our group previously reported that TRIM31 was involved in cerebral ischemic injury by promoting degradation of TIGAR.However,whether TRIM31 is involved in autophagy in cerebral ischemic lesion and the specific mechanism of it remian unclear.In the present study,middle cerebral artery occlusion(MCAO)in mice and oxygen and glucose deprivation and reperfusion(OGD/R)model in cells were used to explore the role and mechanism of TRIM31 involves in cerebral ischemia by regulating autophagy through TLR9 degradation.Firstly,we observed that there was a significant increase in the expression of the autophagic marker LC3 Ⅱ,while p62 expression was decreased in TRIM31 knockout mice after cerebral ischemic injury and TRIM31 deficiency markedly reduced the neurological scores.Consistently,TRIM31 overexpression further decreased the LC3 Ⅱ protein expression in OGD/Rstimulated PC 12 cells and our results verified TRIM31 silence mitigated the decrease of LC3 Ⅱ induced by OGD/R.Meanwhile the TLR9 protein expression was significantly elevated after cerebral ischemic injury and there was a significant increase in the expression of the autophagic marker LC3 Ⅱ,while p62 expression was decreased in TLR9-overexpressed cells induced by OGD/R.Overexpression of TRIM31 could significantly inhibit the up-regulation of TLR9 protein induced by OGD/R stimulation and TRIM31 silence further contributed to the increase of TLR9 induced by OGD/R.We found that the expression of TLR9 was markedly upregulated in TRIM31 dificiency mice and the expression of TLR9 was further upregulated after MCAO.Next,Toll-like receptor 9 inhibitor(E6446)was injected into the ventricle of TRIM31 knockout mice.It was found that TRIM31 further decreased the LC3 Ⅱprotein expression by inhibiting TLR9,while p62 protein expression was increased.In this way,the neurological scores were markedly increased.Furthermore,we identified that TRIM31 binded to TLR9 through its RING domain and conducted ubiquitination modification on TLR9 depending on its own enzyme activity,which negatively regulated the protein level of TLR9.TRIM31 selectively promoted K48linked polyubiquitination of TLR9 and mediated its degradation through the autophagy-lysosomal pathway.In summary,our study clarified that the E3 ubiquitin ligase TRIM31 inhibited autophagy in cerebral ischemia through promoting the degradation of TLR9 by mediating its autophagic lysosomal degradation through K48-linked ubiquitination modification,and aggravated the cerebral ischemic injury.In addition,our study also enriched the regulatory mechanism of TLR9 and revealed that it can play a variety of roles in cerebral ischemia.It provides a new strategy to reveal the pathogenesis of cerebral ischemia.