Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity
Abstract:Background: NMDA receptor and Na ϩ /Ca 2ϩ exchanger are involved in glutamate-induced calcium dysregulation in neurons. Results: CRMP2 interacts with and modulates activity of the NMDA receptor and Na ϩ /Ca 2ϩ exchanger. Conclusion: CRMP2 is involved in regulation of Ca 2ϩ homeostasis in neurons exposed to glutamate. Significance: CRMP2 interaction with NMDA receptor and Na ϩ /Ca 2ϩ exchanger affects their activity and is important for glutamate-induced Ca 2ϩ dysregulation.
“…Testing this hypothesis, we recorded NMDAR activity using multielectrode-array recording of RGCs and found that polyarginine compounds do not interfere with NMDA blockade of lightdriven synaptic responses, confirming that the neuroprotection provided by R(7), C-R(7), and CN2097 is not the result of NMDA-receptor antagonism in the intact retina. Supporting this conclusion, it has been reported that Tat does not induce internalization of the NMDA NR2B subunit and the sodium calcium exchanger (NCX) proteins, whereas the neuroprotective peptide Tat-CBD3, a 15-amino acid peptide from CRMP2 fused to Tat, attenuated NMDAR activity and protected neurons against glutamate-induced Ca 2ϩ dysregulation (52). As mitochondria are key players in signaling neuronal death (35,53,54), and with biotin-C-R(7) peptide localizing to mitochondria (Fig.…”
Background: NMDA receptor hyperactivity results in mitochondrial dysfunction in neurons promoting neurodegenerative disorders. Results: Short polyarginine peptides target mitochondria to promote neuronal survival. Conclusion: Short polyarginine peptides reduce mitochondrial respiration, membrane hyperpolarization, and generation of reactive oxygen species. Significance: Treatment with polyarginine has the potential to minimize neuronal damage resulting from stroke or traumatic brain injury and may be therapeutic to ameliorate multiple sclerosis and Parkinson disease.
“…Testing this hypothesis, we recorded NMDAR activity using multielectrode-array recording of RGCs and found that polyarginine compounds do not interfere with NMDA blockade of lightdriven synaptic responses, confirming that the neuroprotection provided by R(7), C-R(7), and CN2097 is not the result of NMDA-receptor antagonism in the intact retina. Supporting this conclusion, it has been reported that Tat does not induce internalization of the NMDA NR2B subunit and the sodium calcium exchanger (NCX) proteins, whereas the neuroprotective peptide Tat-CBD3, a 15-amino acid peptide from CRMP2 fused to Tat, attenuated NMDAR activity and protected neurons against glutamate-induced Ca 2ϩ dysregulation (52). As mitochondria are key players in signaling neuronal death (35,53,54), and with biotin-C-R(7) peptide localizing to mitochondria (Fig.…”
Background: NMDA receptor hyperactivity results in mitochondrial dysfunction in neurons promoting neurodegenerative disorders. Results: Short polyarginine peptides target mitochondria to promote neuronal survival. Conclusion: Short polyarginine peptides reduce mitochondrial respiration, membrane hyperpolarization, and generation of reactive oxygen species. Significance: Treatment with polyarginine has the potential to minimize neuronal damage resulting from stroke or traumatic brain injury and may be therapeutic to ameliorate multiple sclerosis and Parkinson disease.
“…In many experiments a TAT fused non-arginine containing scrambled peptide (TAT-scramble; TAT-WEAKEMLYFEALVIE; net charge +5) with no amino acid content relationship to the CBD3 peptide was used as a negative control (Brittain et al, 2011a,b;Brustovetsky et al, 2014). In experiments using the TAT-scrambled peptide, negative results similar to the vehicle control were obtained.…”
Section: In Vitro Studiesmentioning
confidence: 92%
“…A further study has also shown that TAT-CBD3 inhibits glutamateand NMDA-induced calcium influx in cultured hippocampal neurons (Brustovetsky et al, 2014), as well as disrupting a CRMP2-NMDA receptor complex interaction, however it did not appear to induce internalisation of the NR2B protein. The same study demonstrated that TAT-CBD3 also inhibits NCX-mediated calcium influx, and that CRMP2 can interact with NCX3 but not with NCX1.…”
Section: In Vitro Studiesmentioning
confidence: 95%
“…There are several lines of evidence based on our findings and those of others that support our endocytosis hypothesis. Arginine-rich peptides, including so called "neuroprotective peptides" fused to TAT have been shown to: i) reduce neuronal calcium influx (Meloni et al, 2015) and interfere with ion channel function (NMDA receptor: Ferrer-Montiel et al, 1988;Tu et al, 2010;Sinai et al, 2010;Brittain et al, 2011b;Brustovetsky et al, 2014, VR1: Planells-Cases et al, 2000, CaV2.2: Brittain et al, 2011a, 2011bFeldman & Khanna, 2013;Brustovetsky et al, 2014;sodium calcium exchanger [NCX], CaV3.3: García-Caballero et al, 2014); ii) cause internalisation of neuronal ion channels (Brustovetsky et al, 2014;Sinai et al, 2010); and iii) require endocytosis as a prerequisite for neuroprotection (Meloni et al, 2015;Vaslin et al, 2011). Interestingly, other TAT-fused peptides have also been shown to interfere with the function of neuronal receptors (D1R-D2R; Pei et al, 2010;PTPσ: Lang et al, 2015).…”
Section: Proposed Neuroprotective Mechanism Of Action Used By Argininmentioning
Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.
“…CRMP2 regulates multiple processes in neurons and was initially discovered to regulate mechanisms of neuronal polarity (9,10). CRMP2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) (11), glycogen synthase kinase 3β (10), Rho-associated protein kinase (12), or the Src-family kinases Fyn (13) and Yes (14) drives its diverse cellular functions, including neurite outgrowth, endocytosis, and ion-channel trafficking (8,(15)(16)(17). Studies of CRMP2 trafficking functions have revealed that CRMP2 facilitates endocytosis of L1-cell adhesion molecule by interacting with the endocytic protein Numb (18) that recruits epidermal growth factor receptor pathway substrate 15 (Eps15), an initiator of clathrin-mediated endocytosis (19).…”
Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrindependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.NaV1.7 sodium channel | trafficking | CRMP2 | SUMOylation | phosphorylation
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