Revisiting Ischemic Acidotoxicity

Acid-sensing ion channels(ASICs)are extracellular proton-gated cation channels.In central nervous system,postsynaptic ASIC1a channel is activated by acidic contents released from presynaptic vesicles during neurotransmission,contributing to synaptic plasticity,learning/memory and fear conditioning.In certain pathological conditions,acid-base balance is disrupted and therefo re excessive protons are generated and accumulated,which is termed acidosis.Acidosis is a common phenomenon and contributes to neuronal injury in kinds of neurological diseases,such as ischemic stroke,multiple sclerosis and Huntington disease.It has been shown that Asic1 gene deletion reduced the infarction volume in middle cerebral artery occlusion(MCAO)mice.Ca²⁺influx via homomeric ASIC1a was considered to be the leading cause of ischemic neuron death.Here,we demonstrated that extracellular protons triggered neuronal necroptosis via ASIC1a,but independent of its ion-conducting function.Acid ic stimulation recruited receptor-interacting protein 1(RIP1)to the ASIC1a C-terminus,causing RIP1phosphorylation and subsequent neuronal necroptosis.Our data further showed that ASIC1a N-terminus exerted neuroprotective effects by intramolecular inhibition of the toxic C-terminus.In this project,we studied the non-conducting function of ASIC1 in ischemic neuronal necroptosis by pharmacological intervention and genetic manipulation in vitro and in vivo.We tried to clarify the role of ASIC1a in ischemic neuronal cell death,screen/identify the ASIC1a-based neuroprotective peptides,which may provide new therapy strategies for stroke.We adopted a modulatory profiling strategy to explore the molecular mechanisms underlying acidic neuronal death.We found that the inhibitor of RIP1 phosphorylation,Nec-1,resulted in a protective effect against acidic neuronal death.MCAO caused RIP1-ASIC1a association and RIP1 phosphorylation in affected brain areas.Deletion of Asic1 gene significantly prevented RIP1 phosphorylation and brain damage,suggesting an important role of ASIC1a-mediated RIP1 phosphorylation in ischemic brain damage.The acidotoxicity effect was recapitulated with a proximal C-terminal peptide of ASIC1a,CP-1,which was assembled with TAT to penetrate cell membrane and induced necroptosis without acid or ischemic treatment.These results indicated that the C-terminus(CT)of ASIC1a played an important role in acid induced RIP1 activation and neuronal necroptosis.Next we found that the N-terminus(N T)of ASIC1a bound to ASIC1a-CT in physiological condition by F?rster resonance energy transfer(FRET).Extracellular acidification activated and led to the conformational changes of ASIC1a,which released the ASIC1a-CT from binding of ASIC1a-NT.Transfection of N T-truncated ASIC1a directly caused CHO cells death even in p H 7.4 condition,which confirmed that N T formed the intramolecular inhibition on C T.To further explore how C T-NT uncoupled,we identified N-ethylmaleimide-sensitive fusion ATPase(NSF)via affinity-purification coupled mass spectrometry.NSF combined with ASIC1a to form NSF-ASIC1a-RIP1complex in MCAO mice,and both Asic1 KO and ASIC1a inhibitor Pc TX1 could disrupt the complex.The death signaling of ASIC1a was inhibited after pharmacological or genetic inhibition of NSF,while channel function of ASIC1a was normal.We identified the specific sequence NT_1-20 of ASIC1a that interacted with NSF,and TAT-NT_1-20disrupted the formation of NSF-ASIC1a-RIP1 complex and RIP1 phosphorylation,prevented neuronal death in culture neurons as well as MCAO mice.All the data confirmed that NSF competed for the binding to N T following exposure to acidosis,which released CT free to interact with RIP1 and induced the ASIC1a-mediated neuronal necroptosis.We also verified the non-conducting function of ASIC1a in Asic1 mutant mice.Mutation of ³²HIF³⁴,which was the key residues in the pore region of ASIC1a,to AAA,caused the loss of channel function.We successfully established HIF mutant mice by CRISPR/Cas9-mediated genome engineering,and confirmed the absence of channel function.In MCAO model,HIF mutant mice exhibited the same level of brain damage as the wild type control.It supported the conclusion that the canonical ion-conducting function of ASIC1a is not the leading cause of ischemic neuronal necroptosis.NSF-ASIC1a-RIP1 complex and RIP1 phosphorylation were increased in MC AO model of HIF mutant mice and both Nec-1 and Pc TX1 reduced the lesion size.The data revealed that the non-conducting function of ASIC1a did not depend on the ion-conducting function of ASIC1a.In conclusion,we revealed the non-conducting function of ASIC1a in ischemic neuronal death and developed a novel neuroprotective ASIC1a-targeted peptide.Our study sheds new light on the role of ASIC1a in neuronal signaling and cell death,and provides potential therapy for neurological diseases.