Ubiquitination Of Glycine Receptors By The Ubiquitin Ligase HUWE1 In Spinal Cord Dorsal Horn

Objective:Glycine receptors(GlyRs)are pentameric proteins that consist ofα(α1-α4)subunits and/orβsubunit.In spinal cord dorsal horn of adult animals,the majority of inhibitory glycinergic neurotransmission is mediated byα1subunit-containing GlyRs.The glycinergic inhibition is reduced by peripheral tissue or nerve injury,which induces pain sensitization.HUWE1(HECT,UBA,WWE domain containing 1)is an E3 ubiquitin ligase that has been shown to ubiquitinate a line of proteins.Previous studies have demonstrated that HUWE1 can regulate the function of immune cells.However,the role of HUWE1 at synapses is unknown as yet.This study was designed to investigate the effects of HUWE1 on glycine receptors and the relationship between HUWE1 and glycinergic disinhibition after peripheral inflammation.Method:Immunocytochemistry was performed to examine the synaptic localization of HUWE1 and the effects of HUWE1 on the intracellular trafficking of glycine receptors in cultured spinal neurons in vitro.The immunocoprecipitation experiments were conducted to examine the interaction of HUWE1 with glycine receptors and the ubiquitination of glycine receptors by HUWE1.The role of HUWE1in glycinergic transmission during inflammatory pain was investigated by using electrophysiological recordings.We also measured the paw withdrawal thresholds(PWTs)and paw withdrawal latenicies(PWLs)to evaluate the contribution of HUWE1 to the allodynia and hyperalgesia induced by Complete Freund’s Adjuvant(CFA).Results:(1)HUWE1 was localized at excitatory and inhibitory synapses of spinal neurons.(2)HUWE1 specifically interacted with glycine receptorα1 subunit,but not with NMDA receptor GluN1 subunit,AMPA receptor GluA1 subunit,or GABA_A receptorβ2 andβ3 subunit.(3)The enhanced neuronal acitivity facilitated the interaction between HUWE1 andα1 by increasing the distribution of HUWE1 at inhibitory synapses.(4)HUWE1 ubiquitinatedα1 in neural activity-dependent manner.(5)HUWE1-mediated ubiquitination promoted the internalization and decreased the surface expression ofα1.(6)Peripheral inflammation increased HUWE1 interaction withα1,through which HUWE1 ubiquitinatedα1 and impaired glycinergic synaptic transmission.(7)Knockdown of HUWE1 expression boosted glycinergic synaptic transmission in spinal cord dorsal horn of inflamed animals.(8)Knockdown of HUWE1 expression effectively alleviated the mechanical allodynia and thermal hyperalgesia induced by CFA.Conclusion:HUWE1 reudced glycinergic synaptic transmission by ubiquitinatingα1 subunit.The inhibition of HUWE1-mediatedα1 ubiquitination was beneficial for the attenuation of pathological pain.