The Research Of The Modulation And Its Mechanism Of CaV2.2by CRMP-2and Its Novel Peptide CBD3

Millions of people were suffered from pathological pain, according to statistics, one thirdof the people in the world were suffering from the persistent or repetitive pain. Neuropathicpain is the hardest problem to solve in various kinds of pathological pain. It is a kind ofpathological pain which is due to the insult or dysfunction of nervous system and is based onthe change of nervous function, structure and neurotransmission. Spontaneous pain,hyperpathia and allodynia are the major performance of the neuropathic pain. The treatmentof the neuropathic pain is thorny, although the fundamental research of pathogenesis areadvancing by leaps and bounds in these years, it is still limited and unsatisfactory in thetreatment. At present, medication treatment is still the main treatment means. Therefore, theresearch hotspot in the future will be the development of new drugs targeted the mechanismof neuropathic pain.A lot of research have suggested that the function of CaV2.2is closely related to the painin sensory neurons. The physiological function of CaV2.2facilitate the release ofneurotransmitter, it can regulate various diverse neurotransmission in which CGRP is the mostinterested. CGRP is a powerful peptide which can cause vasodilatation and have closelyrelation with pain and inflammation. Therefore, a novel medication is required to antagonizethe function of CaV2.2, inhibit the release of CGRP and decrease hypersensitivity of theinjured animal model, consequently achieve the goal to treat the CaV2.2related pain. Myresearch that focused on the CRMP-2discusses the interaction and its mechanism of CRMP-2and CaV2.2. I found a new peptide which is derived from CRMP-2during the research, andfurther study the interaction and its mechanism of CBD3and CaV2.2or NMDARs,illustrating the function of CRMP-2and CBDs in the regulation of neurotransmission, whichis aimed to develop a new research method to treat the CaV2.2related neuropathic pain.Chapter one: The research of the regulation between CRMP-2and CaV2.2Synaptic transmission is coordinated by a litany of protein-protein interactions that relyon the proper localization and function of pre-and post-synaptic Ca2+channels. The axonalguidance/specification collapsin response mediator protein-2(CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+channel (CaV2.2). So far, it isstill unclear that the function of CRMP-2in neurotransmission.The research is aimed to solve several problems:①d etermine if CRMP-2interactsfunctionally and biochemically with CaV2.2;②d etermine if the interaction between CRMP-2and CaV2.2affects the function of CaV2.2;③d etermine if CRMP-2regulate theneurotransmission.The research obtained several results by various study, including biochemical method,immunohistochemical method, electrophysiological method, molecular biological method andanimal behavior test.（1）CRMP-2and CaV2.2are co-localization in the synapse and somesubcellular compartment;（2）CRMP-2and CaV2.2co-exist in a complex;（3）WithinCRMP-2, three regions of interactions were determined: a region in the CRMP-2N-terminus(residues94-166), a region in the middle of the protein (residues212–297), and one near theC-terminus of the CRMP-2protein (residues479-500), proximal to the microtubule bindingdomain. These regions were designated CaV binding domains (CBDs)1–3;（4）WithinCaV2.2, CRMP-2bound two regions on CaV2.2: loop1(L1) and the distal part of theC-terminus (Ct-d);（5）CRMP-2-CaV2.2interaction is activity dependence: The increase ofCa2+influx by CaV2.2enhanced the CRMP-2-CaV2.2interaction. But when theconcentration of Ca2+influx reach100μM, the strength of CRMP-2and CaV2.2interaction isdecreasing according to this;（6）The Ca2+current density was significantly higher inCRMP-2-EGFP-transfected neurons than in EGFP neurons.(P<0.05), and the infection of12DIV hippocampal neurons with CRMP-2shRNA lentivirus for seven days reduced Ca2+currents to2.9±0.4pA/pF (n=9); an~80%reduction compared to CRMP-2–EGFPover-expressing neurons(P<0.05);（7）Immunoblotting with CaV2.2for streptavidin-enrichedcomplexes from biotinylated neurons showed increased CaV2.2surface expression inCRMP-2-EGFP expressing neurons compared to EGFP neurons;（8）Exposing hippocampalneurons transfected with EGFP to30mM KCl stimulated glutamate release by about3.5-foldfrom a basal level. In CRMP-2-EGFP over-expressing neurons,30mM KCl stimulatedglutamate release by about8-fold from a basal level (p<0.001vs. EGFP). This shows thatCRMP-2can augment glutamate release by2.3-fold over control EGFP-transfected cells;（9） CRMP-2S522A and CRMP-2S522D-transfected neurons compared to CRMP-2-KDRWT-transfected neurons, have lower Ca2+influx(P<0.05);（10）The phosphorylation ofCRMP-2by Cdk5leads to a significant increase in the amount of CaV2.2bound to theimmunoprecipitated CRMP-2(P<0.05);（11）To develop a reagent to disrupt the interactionof CRMP-2with the CaV2.2complex in vivo, a series of overlapping15-amino-acid peptideswere synthesized covering the entire length of CRMP-2using a robotic peptide spotter. Thepeptide found to have the highest binding potential for CaV2.2was a15amino acid portion ofthe previously identified CBD3sequence;（12）In order to facilitate the penetrance of theCBD3peptide inside of cell for future studies the CBD3peptide was fused to the transductiondomain of the HIV protein TAT. This yielded the cell penetrating peptides TAT-CBD3;（13）TAT-CBD3affects the CaV2.2to inhibit the Ca2+influx by two means:①T AT-CBD3decreases the CaV2.2current by destroying the CRMP-2-CaV2.2interaction, but not bytargeting CaV2.2；②T AT-CBD3antagonizes the CRMP-2induced Ca2+current enhancement;（14）Slices incubated with TAT-Control displayed a~9fold increase in iCGRP in response toCapsaicin exposure whereas those incubated with TAT-CBD3displayed less than a3foldincrease compared to basal iCGRP levels(P<0.05);（15）A significant increase in PWT wasseen with injection of TAT-CBD3at0.1mg/kg and complete reversal of hypersensitivity wasaccomplished by1mg/kg. No additional increase in PWT was observed when TAT-CBD3was increased to10mg/kg;（16）TAT-CBD3, at doses as high as50mg/kg, showed no effecton the animals’ ability to stay upright on the rotating rod at1h or up to7days later.;（17）Following TAT-CBD3injection animals were tested in the Morris water maze at3h,1,2, and3days. Animals showed no significant deficit in latency to reach the platform at any of thesetime points.Main conclusion:①CRMP-2is the important regulator to CaV2.2：CRMP-2canfacilitate to transfer CaV2.2to membrane, lead to the increase of CaV2.2expression in thesurface membrane, then cause the enhancement of the CaV2.2current;②Theoverexpression of CRMP-2can facilitate the CaV2.2induced neurotransmitter release;③The phosphorylation of CRMP-2by Cdk5at Ser-522is the important determinant to theregulation of CaV2.2;④T AT-CBD3is a novel antagonist of CaV2.2, it can inhibit the release of CGRP, and decrease the hypersensitivity of the injured animal models, therefore itcan be used to treat the CaV2.2related neuropathic pain and so on, additionally TAT-CBD3will not cause a sever sedation and deficient in the function of motor and coordinate as wellas the memory extracted.Chapter2: The research of the neuroprotection and its mechanism of TAT-CBD3Unchecked Ca2+influx via N-methyl-D-aspartate receptors (NMDARs) has been linkedto activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2was suggested to affect NMDAR trafficking and possibly involved in neuronal survivalfollowing excitotoxicity. Based upon these studies, I hypothesized that a peptide fromCRMP2could preserve neurons in the face of excitotoxic challenges.In order to determine the neuroprotection and its mechanism of TAT-CBD3which isderived from CRMP-2and fused to cell penetrating peptide TAT, I did a bunch of studies bythe similar methods as mentioned above, and obtain several results:（1）At24hours postglutamate induced stimulation, neurons exposed to TAT-CBD3had~70%less cleavedCRMP-2compared to neurons exposed to TAT-Control(P<0.05); TAT-CBD3does not altercleavage of CRMP-2in response to Ionomycin；TAT-CBD3does not alter in vitro cleavage ofCRMP-2and activation of calpain;（2）Neurons pre-treated for10min with10μMTAT-CBD3and then stimulated with Glu/Ser completely survived the excitotoxic treatment.Significant neuroprotection by TAT-CBD3was evident at3and1μM but not at0.1μM(P<0.05); To test if TAT-CBD3is neuroprotective following stimulation, neurons weretreated with10μM TAT-CBD3for10minutes at0.5,1,2,3, and6hours after Glu/Serstimulation and cell viability measured as described. Except for modest, but statisticallysignificant, neuroprotection at0.5hour following stimulation, TAT-CBD3was not effective atlater times(P<0.05)（；3）CRMP-2shRNA lentiviral–transduced neurons exhibited a significantincrease in cell viability following stimulation compared to scramble shRNA treated neuronswhen pre-treated with TAT-Control(p<0.05). Complete neuroprotection was observed inneurons transduced with CRMP-2shRNA lentivirus and pre-treated with TAT-CBD3prior toexcitotoxic challenge. That neurons transduced with scramble shRNA lentivirus did notexhibit complete neuroprotection with TAT-CBD3;（4）Lysates were then made24hr following stimulation and CRMP-2cleavage was assessed by immunoblotting. Treatmentwith ω-CTX with or without stimulation by Glu/Ser had no effect on CRMP-2cleavage；Theblockade of CaV2.2with ω-CTX did not significantly alter neuronal survival of neuronsfollowing Glu/Ser；（5）Both vehicle and TAT-Control displayed similar increases in [Ca2+]cduring the prolonged exposure to glutamate. In contrast, TAT-CBD3showed a significantdecrease in [Ca2+]ccompared to vehicle and TAT-Control（P<0.05）. The changes in [Ca2+]cfollowing glutamate stimulation, represented by average area under the curve (AUC), wasdecreased by78%and75%compared to that observed in TAT-Control and vehicle-treatedneurons, respectively（P<0.05）;（6）A sharp increase in [Ca2+]cwas observed when NMDAis applied, which returned to baseline upon NMDA removal. When vehicle (DMSO) wasapplied during the interim between the three NMDA stimulations, no change in [Ca2+]camplitude was observed. However, when neurons were treated with10μM TAT-CBD3duringthis interim period, the second and third responses to NMDA were strongly attenuated. Theratio of the2nd to1st NMDA-induced Ca2+peak (P2/P1) for vehicle treated neurons was1.05±0.09whereas incubation with10μM TAT-CBD3reduced the P2/P1ratio to0.25±0.03. Theratio of the3rd to1st Ca2+peak (P3/P1) for TAT-CBD3neurons was0.33±0.03.Co-application of TAT-CBD3with100μM AP-5led to a P2/P1ratio of0.19±0.03and aP3/P1of0.93±0.08;（7）Neurons were transfected with CRMP-2siRNA+GFP at5DIVand then Ca2+imaging was performed using a two-pulse NMDA protocol at7DIV. Neuronstreated with CRMP-2siRNA exhibited reduced inhibition of NMDAR-mediated Ca2+-influxby TAT-CBD3(P2/P1=0.60±0.09) vs. untransfected controls (P2/P1=0.29±0.06, p <0.05);（8）I employed the use of the NR2B and NR2A specific blockers Ifenprodil and Peaqxrespectively to accomplish this goal. Cortical neurons were treated for10minutes prior tostimulation and throughout stimulation with200μM glutamate and20μM glycine for30min.At7DIV it was found that Ifenprodil, but not Peaqx, was able to completely preventGlu/Gly-induced toxicity（P<0.05）;（9）Incubation of neurons with10μM TAT-Controlshowed no significant change in fluorescence at20minutes compared to vehicle. In contrast,incubation with10μM TAT-CBD3caused a~60%reduction in NR2B-SEPfluorescence(P<0.05）;（10）Concomitant addition of the NMDAR antagonist MK801(50μM) with10μM TAT-CBD3completely prevented the TAT-CBD3-induced reduction inNR2B-SEP fluorescence observed at20minutes following peptide/drug application.Treatment with50μM MK801alone had no effect on NR2B-SEP fluorescence;（11）NMDAR currents were recorded from rat hippocampal neuron somas (DIV7-11) usingwhole-cell voltage-clamp electrophysiology by stimulating with50μM NMDA for2secondswith a30second interval between stimulations. Following5minutes of stableNMDA-stimulated responses neurons were perfused with10μM TAT-CBD3. TAT-CBD3induced rapid and strong inhibition of NMDA currents with~70%block after5minutes.Washout with bath solution lacking TAT-CBD3led to partial recovery of NMDA currentsafter10minutes.Main conclusion:①T AT-CBD3inhibits the α-spectrin and CRMP-2calpain cleavage bydecreasing the glutamate-induced Ca2+influx;②CRMP-2knockdown is neuroprotective, andnot only involved in glutamate-induced exitotoxcity, but also effectively targetes to preventexitotoxcity-induced cell death;③T AT-CBD3is a novel neuroprotective peptide, it leads tothe decrease of glutamate-induced DCD and NMDA-stimulated Ca2+influx by inhibiting theNMDARs or inducing NMDAR in dendrite spine internalization, then it can be used to treatthe chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.