CRMP2-derived Peptide ST2-104 Protects SH-SY5Y Cells Against A?_25-35-induced Damage By Inhibiting The CRMP2-NMDAR2B Signalling Pathway

Alzheimer's disease?AD?is an age-related progressive neurodegenerative disorder that is characterised by learning,spatial memory deficits and cognitive dysfunction.The main pathological changes include a decrease in synapses and neurons,the abnormal extracellular amyloid beta protein?A??accumulation and intracellular neurofibrillary tangles?NFTs?in the cortex and hippocampus.Current treatments cannot effectively improve the manifestation and block the progression of the disease.Without an effective therapy,it is true that the number of patients will double by the year 2050.Therefore,creating a new therapeutic strategy is meaningful for the treatment of AD.Collapsin response mediator protein 2?CRMP2?,traditionally regarded as an axon/dendrite growth and guidance protein,has played an important role in regulation of both post-and pre-synaptic Ca²⁺channels such as N-methyl-D-aspartate receptors?NMDARs?.CRMP2 is broadly distributed in cortex and hippocampus,which is highly phosphorylated by CDK5 and GSK3?in an AD brain.It was shown that the phosphorylation of CRMP2 can strengthen the interaction between CRMP2 and NMDARs,which causes excessive NMDARs activation,whereas blocking the combination of CRMP2 and NMDARs or knocking out CRMP2 could protect neurons from injury in cerebral ischaemia and brain trauma.The Ca²⁺channel-binding domain 3?CBD3?peptide derived from CRMP2 has recently emerged as a Ca²⁺channel blocker,which suppressed neuropathic pain in a spared nerve injury?SNI?model when linked to the transduction domain of HIV TAT protein and reduced neuronal death in a middle cerebral artery occlusion model and a traumatic brain injury?TBI?model.Here,we sought to research the neuroprotective effects and the biochemical mechanisms of ST2-104?a nona-arginine-conjugated CBD3 peptide,sequence?N?C?:RRRRRRRRRARSRLAELRGVPRGL?against A?_25-35-induced neurotoxicity in SH-SY5Y cells.Methods:Firstly,to test this neuroprotective effect,we exposed the SH-SY5Y cells to toxic concentrations of A?_25-35 peptide,and measured cell survival by MTT assay,damage by LDH assay and apoptosis by Hoechst 33258 assay after a preincubation with ST2-104.Western Blot was used to detect the protein expression of CRMP2,pCRMP2,Bax,Bcl-2.Co-Immunoprecipitation was used to detect the interaction of pCRMP2/NMDAR2B.Flow Cytometry was used to detect the intracellular free Ca²⁺.Secondly,to further explore the mechanism of the neuroprotective effect of ST2-104 and the key role of CRMP2 in AD,we induced CRMP2 over-expression or knockdown by lentiviral vector infection.Control cells were infected with gene GFP-encoded lentiviral vector or scramble shRNA lentiviral vector.Afterwards,the expression of CRMP2 was detected by Western Blot assay.We evaluated this neuroprotective effect by the ways described above.In addition,Whole-cell voltage-clamp was used to detect the NMDARs Ca²⁺currents.Results:Compared with the control group,A?_25-35?model group?significantly decreased the cell viability,increased the LDH release and induced apoptosis.Western blot showed that A?_25-35 significantly enhance the expression of pCRMP2 and Bax,but decreased the expression of Bcl-2.Co-IP showed that A?_25-35 significantly enhance the interaction of pCRMP2/NMDAR2B.FCM showed that A?_25-35significantly increased the concentration of intracellular free Ca²⁺.Compared with the model group,ST2-104?ST2-104 group?significantly increased the cell viability,decreased the LDH release and inhibited apoptosis.Western blot showed that A?_25-35significantly decreased the expression of pCRMP2 and Bax,but enhance the expression of Bcl-2.Co-IP showed that A?_25-35 significantly inhibited the interaction of pCRMP2/NMDAR2B.FCM showed that A?_25-35 significantly decreased the concentration of intracellular free Ca²⁺.Western blot and fluorescence assay showed that Lentiviral vectors effectively infected SH-SY5Y cells.All assays described above showed that CRMP2 over-expression significantly increased the sensibility of A?_25-35toxicity,but CRMP2 knockdown significantly decreased the sensibility of A?_25-35toxicity.ST2-104 significantly protected SH-SY5Y cells against A?25-35-induced damage.Conclusion:We demonstrated that ST2-104 relieved A?_25-35-induced neurotoxicity in SH-SY5Y cells by suppressing the expression of pCRMP2,and attenuated the level of intracellular Ca²⁺by disrupting the interaction between pCRMP2/NMDAR2B.CRMP2/NMDAR interactions may play an important role in A?_25-35-induced neurotoxicity in SH-SY5Y cells and that the modulation of NMDARs by CRMP2 can have a protective effect on neurons.Taken together,these findings support ST2-104 as a novel neuroprotective agent,which potentially represents a novel direction for a therapeutics targeting channel in AD.