The Autophagy-related Protein TMEM59 Interacts With TREM2 And Participates In Neuroinflammation

Autophagy is a common mechanism underlying cellular homeostasis in eukaryotes,and plays a vital role in regulating development and tissue homeostasis.Moreover,autophagy can reduce inflammation by regulating pathogen clearance,antigen presentation,and innate and adaptive immune responses.The single transmembrane protein TMEM59 has been found to be associated with autophagy,affect cognitive function,and participate in neuroinflammation regulation;however,the underlying mechanism remains largely elusive.TREM2 is a receptor specifically expressed in microglia of the central nervous system and plays an important role in maintaining normal microglial function.Mutations in TREM2 are risk factors for the pathogenesis of Alzheimer’s disease,a devastating disorder with severe cognitive dysfuntion.Our laboratory previously found that TMEM59 interacted with TREM2.But the biological significance of such an interaction is unknown.Therefore,in the present study,we further determined the exact role of TMEM59 in autophagy,investigated the significance of the TMEM59-TREM2 interaction,and explored the function of TMEM59 in neuroinflammation.We first found that overexpression of full-length TMEM59 and TMEM59 carboxyl-terminal fragment(CTF),but not TMEM59 amino-terminal fragment(NTF)increased the levels of LC3B-Ⅱ,a classical autophagy marker,and promoted autophagy flux.TMEM59 overexpression had no additional effect on LC3B-Ⅱ level increase upon stimulation by the autophagy activator rapamycin,whereas its overexpression further promoted LC3B-Ⅱ level increase caused by inhibiting the autophagy flux by bafilomycin A and hydroxycholoroquine.In addition,we found that both full-length TMEM59 and TMEM59-CTF interacted with autophagy-related proteins ATG5,ATG16L1,and LC3B.Together,these results suggest that TMEM59 induces autophagy at downstream of the autophagy initiation but upstream of the autophagolysosome formation,and probably through interacting with ATG5,ATG16L1 and LC3B.Through constructing various truncated forms and by applying coimmunoprecipitation,we identified that TREM2 interacted with TMEM59 probably through their transmembrane domains.Interestingly,we found that overexpression of TREM2 reduced TMEM59 protein levels through promoting TMEM59 degradation,whereas overexpression of TMEM59 had no effect on TREM2 levels.We also confirmed that loss of TREM2 in microglia resulted in impaired survival,proliferation,migration,and phagocytosis,as well as abnormal autophagy and metabolism.Importantly,we found that downregulation of TMEM59 in Trem2 knockout(KO)microglia largely reversed these damages.At the same time,we found that downregulation of TMEM59 inhibited the NFκB-NLRP3-GSDMD signaling,reduced the expression of pro-inflammatory factors,and increased the expression of anti-inflammatory factors.Treatments with lipopolysaccharide(LPS),kanic acid,and starvation reduced TMEM59 expression.While downregulation of TMEM59 alleviated LPS-induced inflammation.We further explored the potential role of TMEM59 in the gram-positive bacteria Staphylococcus aureus-induced meningitis.We found that loss of Tmem59 reduced inflammatory responses,promoted microglial activation and phagocytosis,increased bacterial clearance,and improved the clinical performance and survival rate of mice with Staphylococcus aureus-induced meningitis.In summary,our study further confirms the promoting activity of TMEM59 in autophagy,demonstrates the importance of TMEM59 homeostasis in maintaining TREM2-mediated microglial activities,and reveals the neuroinflammation attenuation upon TMEM59 deficiency.These findings provide important clues for future work dissecting the exact role of TMEM59 and autophagy in microglial functions and the potential of TMEM59 as a therapeutic target for neuroinflammatory diseases and Alzheimer’s disease.