PHLDA1 Regulates Microglial Activation And Protects Dopaminergic Neurons Against Inflammation-induced Cell Death

Aims: Microglial activation-mediated neuroinflammation plays an important role in the progression of neurodegerative diseases.A better understanding on the role of inflammation in Parkinson's disease(PD)will provide new insights into the pathological processes and help to establish effective therapeutic strategies.In the present study,we explored the roles of PHLDA1(Pleckstrin homology-like domain family A member 1),in microglial function and neuronal protection.We found that PHLDA1 deficiency caused several phenotypic changes in macroglia.We further to reveal the roles of PHLDA1 in the microglia-mediated neuroinflammation and elicit the molecular mechanisms underlied the effect.Methods:(1)The expression of PHLDA1 in response to TLRs stimuli in BV-2 microglia was assessed by q PCR and Western blotting.(2)BV-2 cells with PHLDA1 knockdown were expoed to LPS,then the proinflammatory genes expression were determined by q PCR and Western blotting.The NF-?B and MAPK pathways were detected to reveal the underlying mechanisms of PHLDA1 regulated microglial activation.(3)HEK293T cells were transfected with TRAF6-Flag and PHLDA1-Myc plasmids,the interaction between PHLDA1 and TRAF6 was determined by exogenous co-immunoprecipitation.Immunoprecipitation analysis was used for TRAF6 K63 ubiquitination.(4)To determine the effect of PHLDA1 deletion in the substantia nigra on motor dysfunction,mice were previously infected with AAV-NC or AAV-sh PHLDA1 in the SNc in PD model,then were administered with behavior assessment.Results:(1)PHLDA1 m RNA and protein levels were increased in response to inflammatory stimuli in microglia.(2)PHLDA1 deficiency inhibits LPS-induced proinflammatory genes expression and attenuates the LPS-induced NF-?B inflammatory pathway activation.(3)PHLDA1 directly interacts with TRAF6 promotes TRAF6-mediated NF-?B activation.PHLDA1 deficiency attenuates K63 ubiquitination of TRAF6.(4)PHLDA1 deficiency ameliorates MPTP-induced motor deficits,and significantly ameliorates MPTP-induced dopaminergic neuronal loss and microglial activation in mouse PD model.Conclusions: PHLDA1 plays an important role in the microglial inflammatory activation.The underlying mechanisms may be associated with PHLDA1-attenuated K63 ubiquitination of TRAF6.PHLDA1 deficiency inhibits neuroinflammation that contributes to dopaminergic neuronal survival.Thus,PHLDA1 may be a potential target for PD.