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The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10−7 M, 10−8 M, and 10−9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10−7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

The mammalian cerebral cortex is characterized by a 6-layer structure, and proper neuronal migration is critical for its formation. Cyclin-dependent kinase 5 (Cdk5) has been shown to be a critical kinase for neuronal migration. Several Cdk5 substrates have been suggested to be involved in ordered neuronal migration. However, in vivo loss-of-function studies on the function of Cdk5 phosphorylation substrates in neuronal migration in the developing cerebral cortex have not been reported. In this study, we demonstrated that Cdk5-mediated phosphorylation of collapsing mediator protein (CRMP) 2 is critical for neuronal migration in the developing cerebral cortex with redundant functions of CRMP1 and CRMP4. The cerebral cortices of triple-mutant CRMP1 knock-out (KO); CRMP2 knock-in (KI)/KI; and CRMP4 KO mice showed disturbed positioning of layers II–V neurons in the cerebral cortex. Further experiments using bromodeoxyuridine birthdate-labeling and in utero electroporation implicated radial migration defects in cortical neurons. Ectopic neurons were detected around the CA1 region and dentate gyrus in CRMP1 KO; CRMP2 KI/KI; and CRMP4 KO mice. These results suggest the importance of CRMP2 phosphorylation by Cdk5 and redundancy of CRMP1 and CRMP4 in proper neuronal migration in the developing cerebral cortex and hippocampus.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow towards, and connect with, six extraocular muscles in a stereotyped pattern, in order to control eye movements. The signalling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that manipulation of α2-chn signalling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signalling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins - collapsin response mediator protein 2 (CRMP2), (encoded by the gene dpysl2), stathmin1 and stathmin 2 - bind to α2-CHN. dpysl2, stathmin1 and especially stathmin2 are expressed by ocular motor neurons. We find that manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases these phenotypes were reminiscent of DRS. chn1 knockdown phenotypes were rescued by overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signalling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance, and to control eye movements.Significance statementThe precise control of eye movement is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unravelled how alpha2-chimaerin co-ordinates axon guidance of the ocular motor system in animal models. In this paper, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signalling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the aetiology of eye movement disorders in humans.

Alzheimer's disease (AD) is a type of progressive neurodegenerative disease, and amyloid β-protein 42 (Aβ42) serves an important role in the pathological process of development of AD. Paired immunoglobulin-like receptor B (PirB) is a functional receptor for myelin inhibitors of neuron regeneration in the CNS, and it has also been identified to function as a high-affinity receptor for Aβ. Here, we used a phage display to identify a specific PirB antagonist peptide 11(PAP11, PFRLQLS), which could reverse Aβ42-induced neurotoxicity and promote neurite outgrowth in vitro. Immunofluorescence analysis showed that PAP11 colocalized with PirB on the membrane of cortical neurons. Horseradish peroxidase-streptavidin-biotin assay further proved that PAP11 directly binds to PirB and the dissociation constant (Kd) was 0.128μM. PAP11 functionally antagonized the neurite outgrowth inhibitory effect induced by Aβ42 in cortical neurons, and the underlying mechanism was associated with a PirB-ROCK2/CRMP2 signaling pathway. The novel PirB antagonist peptide PAP11 may be a promising candidate therapeutic agent for the treatment of AD and other neurodegenerative diseases. KEY POINTS: • PAP11 was the first PirB antagonist peptide screened by phage display technology. • PAP11 could protect neurons by blocking the binding of Aβ42 and PirB. • PAP11 reverse inhibitory effect of neurite outgrowth through ROCK2/CRMP2 pathway.

Deubiquitinating enzymes are increasingly recognized to play important roles in cancer, with many acting as oncogenes or tumor suppressors. In this study, we employed a bioinformatics approach to screen for enzymes from this family involved in cancer and found USP24 as a potent predictor of poor outcomes in neuroblastoma, an aggressive childhood cancer. USP24 resides in a region commonly deleted in neuroblastoma, yet was independently associated with poor outcomes in this disease. Deletion of Usp24 in a murine model resulted in degradation of collapsin response mediator protein 2 (CRMP2), a regulator of axon growth, guidance, and neuronal polarity. Cells lacking USP24 had significant increases in spindle defects, chromosome missegregation, and aneuploidy, phenotypes that were rescued by the restoration of CRMP2. USP24 prevented aneuploidy by maintaining spindle-associated CRMP2, which is required for mitotic accuracy. Our findings further indicate that USP24 is a tumor suppressor that may play an important role in the pathogenesis of neuroblastoma. Significance: This study identifies the chromosome instability gene USP24 as frequently deleted in neuroblastoma and provides important insight into the pathogenesis of this aggressive childhood cancer.

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function. Electronic supplementary material The online version of this article (10.1007/s12035-020-02038-6) contains supplementary material, which is available to authorized users.

Autism spectrum disorder (ASD) is a heterogeneous condition with multiple etiologies and risk factors - both genetic and environmental. Recent data demonstrate that the immune system plays an important role in prenatal brain development. Deregulation of the immune system during embryonic development can lead to neurodevelopmental changes resulting in ASD. One of the potential etiologic factors in the development of ASD has been identified as the presence of maternal autoantibodies targeting fetal brain proteins. The type of ASD associated with the presence of maternal autoantibodies has been referred to as maternal antibodies related to ASD (MAR ASD). The link between maternal autoantibodies and ASD has been demonstrated in both clinical studies and animal models, but the exact mechanism of their action in the pathogenesis of ASD has not been clarified yet. Several protein targets of ASD-related maternal autoantibodies have been identified. Here, we discuss the role of microtubule-associated proteins of the collapsin response mediator protein (CRMP) family in neurodevelopment and ASD. CRMPs have been shown to integrate multiple signaling cascades regulating neuron growth, guidance or migration. Their targeting by maternal autoantibodies could change CRMP levels or distribution in the developing nervous system, leading to defects in axon growth/guidance, cortical migration, or dendritic projection, which could play an etiological role in ASD development. In addition, we discuss the future possibilities of MAR ASD treatment.

Plexin family proteins mediate semaphorin signalling during dendritic arbour development. However, the role of PlexinA3 in the growth of dendrites of cultured cerebellar granule neurons (CGNs) is not known. We found that PlexinA3 colocalizes with CRMP2 (collapsin response mediator protein 2) in dendritic shafts. Immunoprecipitation and glutathione transferase pulldown assays showed that the intracellular Ras-binding domain of PlexinA3 directly interacts with CRMP2. PlexinA3 was necessary and sufficient for the growth of CGN dendrites, as genetic knockdown of PlexinA3 reduced but its overexpression increased dendritic lengths and dendritic tip numbers. These increases were enhanced with CRMP2 overexpression and abolished with CRMP2 knockdown, indicating that CRMP2 is the downstream effector. Furthermore, PlexinA3/CRMP2 signalling contributed to Sema3A-controlled dendritic growth. Together, these data identify a novel PlexinA3/CRMP2 pathway in semaphorin-regulated growth of cultured CGN dendrites.

Calcium dysregulation is a key pathological event in Alzheimer’s disease (AD). In studying approaches to mitigate this calcium overload, we identified the collapsin response mediator protein 2 (CRMP2), an axonal guidance protein that participates in synapse dynamics by interacting with and regulating activity of N-methyl-D-aspartate receptors (NMDARs). We further identified a 15 amino acid peptide from CRMP2 (designated CBD3, for calcium-binding domain 3), that reduced NMDAR-mediated Ca2+ influx in cultured neurons and post-synaptic NMDAR-mediated currents in cortical slices. Whether targeting CRMP2 could be therapeutically beneficial in AD is unknown. Here, using CBD3, we tested the utility of this approach. Employing the APP/PS1 mouse model of AD which demonstrates robust pathophysiology including Aβ1–42 deposition, altered tau levels, and diminished cognitive functions, we asked if overexpression of CBD3 could rescue these events. CBD3 was engineered into an adeno-associated vector and nasally delivered into APP/PS1 mice and then biochemical (immunohistochemistry, immunoblotting), cellular (TUNEL apoptosis assays), and behavioral (Morris water maze test) assessments were performed. APP/PS1 mice administered adeno-associated virus (AAV, serotype 2) harboring CBD3 demonstrated: (i) reduced levels of Aβ1–42 and phosphorylated-tau (a marker of AD progression), (ii) reduced apoptosis in the hippocampus, and (iii) reduced cognitive decline compared with APP/PS1 mice or APP/PS1 administered a control virus. These results provide an instructive example of utilizing a peptide-based approach to unravel protein-protein interactions that are necessary for AD pathology and demonstrate the therapeutic potential of CRMP2 as a novel protein player in AD.

Mechanistic studies principally focusing on primary afferent nociceptive neurons uncovered the upregulation of collapsin response mediator protein 2 (CRMP2)-a dual trafficking regulator of N-type voltage-gated calcium (Cav2.2) as well as Nav1.7 voltage-gated sodium channels-as a potential determinant of neuropathic pain. Whether CRMP2 contributes to aberrant excitatory synaptic transmission underlying neuropathic pain processing after peripheral nerve injury is unknown. Here, we interrogated CRMP2's role in synaptic transmission and in the initiation or maintenance of chronic pain. In rats, short-interfering RNA-mediated knockdown of CRMP2 in the spinal cord reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents, but not spontaneous inhibitory postsynaptic currents, recorded from superficial dorsal horn neurons in acute spinal cord slices. No effect was observed on miniature excitatory postsynaptic currents and inhibitory postsynaptic currents. In a complementary targeted approach, conditional knockout of CRMP2 from mouse neurons using a calcium/calmodulin-dependent protein kinase II alpha promoter to drive Cre recombinase expression reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents, but not miniature excitatory SCss. Conditional knockout of CRMP2 from mouse astrocytes using a glial fibrillary acidic protein promoter had no effect on synaptic transmission. Conditional knockout of CRMP2 in neurons reversed established mechanical allodynia induced by a spared nerve injury in both male and female mice. In addition, the development of spared nerve injury-induced allodynia was also prevented in these mice. Our data strongly suggest that CRMP2 is a key regulator of glutamatergic neurotransmission driving pain signaling and that it contributes to the transition of physiological pain into pathological pain.

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

We have previously reported that the microtubule-associated collapsin response mediator protein 2 (CRMP2) is necessary for the expression of chronic pain. CRMP2 achieves this control of nociceptive signaling by virtue of its ability to regulate voltage-gated calcium and sodium channels. To date, however, no drugs exist that target CRMP2. Recently, the small molecule edonerpic maleate (1 -{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer’s disease was reported to be a novel CRMP2 binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo. Here we sought to determine the mechanism of action of edonerpic maleate and test its possible effect in a rodent model of chronic pain. We observed: (i) no binding between human CRMP2 and edonerpic maleate; (ii) edonerpic maleate had no effect on CRMP2 expression and phosphorylation in dorsal root ganglion (DRG) neurons; (iii) edonerpic maleate-decreased calcium but increased sodium current density in DRG neurons; and (iv) edonerpic maleate was ineffective in reversing post-surgical allodynia in male and female mice. Thus, while CRMP2 inhibiting compounds remain a viable strategy for developing new mechanism-based pain inhibitors, edonerpic maleate is an unlikely candidate.

Knockout of phosphatase and tensin homolog (PTEN−/−) is neuroprotective and promotes axon regeneration in mature neurons. Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN−/− also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3S/A knockin mice significantly compromised PTEN−/−-mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. Gsk3S/A knockin or CRMP2 inhibition also decreased PTEN−/− mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN−/− mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration., Loss of the PTEN protein, an event that activates PI3K/AKT signaling, in neurons enables axon regeneration. Now Leibinger et al show in retinal ganglion cells and during optic nerve regeneration in mice, that this is mediated by inactivation of the AKT substrate GSK3, which in turn releases axonal CRMP2 from GSK3-mediatated inhibition.

Cocaine addiction is a major health concern with limited effective treatment options. A better understanding of mechanisms underlying relapse may help inform the development of new pharmacotherapies. Emerging evidence suggests that collapsin response mediator protein 2 (CRMP2) regulates presynaptic excitatory neurotransmission and contributes to pathological changes during diseases, such as neuropathic pain and substance use disorders. We examined the role of CRMP2 and its interactions with a known binding partner, CaV2.2, in cocaine-seeking behavior. We employed the rodent self-administration model of relapse to drug-seeking and focused on the prefrontal cortex (PFC) for its well-established role in reinstatement behaviors. Our results indicated that repeated cocaine self-administration resulted in a dynamic and persistent alteration in the PFC expression of CRMP2 and its binding partner, the CaV2.2 (N-type) voltage-gated calcium channel. Following cocaine self-administration and extinction training, the expression of both CRMP2 and CaV2.2 was reduced relative to Yoked saline controls. By contrast, cued-reinstatement potentiated CRMP2 expression and increased CaV2.2 expression above extinction levels. Lastly, we utilized the recently developed peptide myr-TAT-CBD3 to disrupt the interaction between CRMP2 and CaV2.2 in vivo. We assessed the reinstatement behavior after infusing this peptide directly into the medial PFC and found that it decreased cue-induced reinstatement of cocaine seeking. Taken together, these data suggest that neuroadaptations in the CRMP2/CaV2.2 signaling cascade in the PFC can facilitate drug seeking behavior. Targeting such interactions has implications for the treatment of cocaine relapse behavior.

Dexmedetomidine has been reported to ameliorate propofol-induced neurotoxicity in neonatal animals. However, the underlying mechanism is still undetermined. Glycogen synthase kinase-3β (GSK-3β), cycline-dependent kinase-5 (CDK5), and Rho-kinase (RhoA) pathways play critical roles in neuronal development. The present study is to investigate whether GSK-3β, CDK5, and RhoA pathways are involved in the neuroprotection of dexmedetomidine. Seven-day-old (P7) Sprague Dawley rats were anesthetized with propofol for 6 h. Dexmedetomidine at various concentrations were administered before propofol exposure. Neuroapoptosis, the neuronal proliferation, and the level of neurotransmitter in the hippocampus were evaluated. The effects of GSK-3β inhibitor SB415286, CDK5 inhibitor roscovitine, or RhoA inhibitor Y276321 on propofol-induced neurotoxicity were assessed. Propofol-induced apoptosis in the hippocampal neurons and astrocytes, inhibited neuronal proliferation in the dentate gyrus region, down-regulated the level of γ-aminobutyric acid and glutamate in the hippocampus, and impaired long-term cognitive function. These harmful effects were reduced by pretreatment with 50 μg·kg−1 dexmedetomidine. Moreover, propofol-activated GSK-3β and CDK5 pathways, but not RhoA pathway, by reducing the phosphorylation of GSK-3β (ser 9), increasing the expression of CDK5 activator P25 and increasing the phosphorylation of their target sites on collapsin response mediator protein 2 (CRMP2) shortly after exposure. These effects were reversed by pretreatment with 50 μg·kg−1 dexmedetomidine. Furthermore, SB415286 and roscovitine, not Y276321, attenuated the propofol-induced neuroapoptosis, brain cell proliferation inhibition, γ-aminobutyric acid and glutamate downregulation, and learning and memory dysfunction. Our results indicate that dexmedetomidine reduces propofol-induced neurotoxicity and neurocognitive impairment via inhibiting activation of GSK-3β/CRMP2 and CDK5/CRMP2 pathways in the hippocampus of neonatal rats.

The peripherally expressed voltage-gated sodium NaV1.7 (gene SCN9A) channel boosts small stimuli to initiate firing of pain-signaling dorsal root ganglia (DRG) neurons and facilitates neurotransmitter release at the first synapse within the spinal cord. Mutations in SCN9A produce distinct human pain syndromes. Widely acknowledged as a "gatekeeper" of pain, NaV1.7 has been the focus of intense investigation but, to date, no NaV1.7-selective drugs have reached the clinic. Elegant crystallographic studies have demonstrated the potential of designing highly potent and selective NaV1.7 compounds but their therapeutic value remains untested. Transcriptional silencing of NaV1.7 by a naturally expressed antisense transcript has been reported in rodents and humans but whether this represents a viable opportunity for designing NaV1.7 therapeutics is currently unknown. The demonstration that loss of NaV1.7 function is associated with upregulation of endogenous opioids and potentiation of mu- and delta-opioid receptor activities, suggests that targeting only NaV1.7 may be insufficient for analgesia. However, the link between opioid-dependent analgesic mechanisms and function of sodium channels and intracellular sodium-dependent signaling remains controversial. Thus, additional new targets - regulators, modulators - are needed. In this context, we mine the literature for the known interactome of NaV1.7 with a focus on protein interactors that affect the channel's trafficking or link it to opioid signaling. As a case study, we present antinociceptive evidence of allosteric regulation of NaV1.7 by the cytosolic collapsin response mediator protein 2 (CRMP2). Throughout discussions of these possible new targets, we offer thoughts on the therapeutic implications of modulating NaV1.7 function in chronic pain.

Collapsin response mediator protein 2 (CRMP2),by regulating voltage-gated calcium channel activity, is a crucial regulator of neuronal excitability. Hyperphosphorylation of CRMP2 has been reported in brains of Alzheimer's disease (AD) patients and other neurodegenerative diseases. CRMP2 acting on N-methyl-d-aspartate receptors (NMDARs) may contribute to AD pathology. A short peptide from CRMP2, designated the Ca2+ channel-binding domain 3 (CBD3) peptide, has recently emerged as a Ca2+ channel blocker that exerts neuroprotective effects in traumatic brain injury and cerebral ischemia by disrupting pCRMP2/NMDAR interaction to inhibit calcium influx. ST2-104, a nona-arginine (R9)-conjugated CBD3 peptide derived from CRMP2, exerts a beneficial effect on neuropathic pain; however, the effect of ST2-104 on AD and its mechanism of action have not been studied. In this study we investigated the effects of ST2-104 on SH-SY5Y neuroblastoma cells stimulated by Aβ25-35. To induce neurotoxicity, SH-SY5Y cells were incubated with Aβ25-35, the shortest toxic fragment of Aβ. CRMP2 expression was manipulated by knockdown or overexpression of CRMP2 before ST2-104 treatment to further explore if the pCRMP2/NMDAR2B signaling pathway is involved in the action of the ST2-104 peptide. The results show that ST2-104 significantly enhanced cell viability, inhibited cell apoptosis, decreased LDH release, suppressed the expression of the pCRMP2 protein, disrupted pCRMP2/NMDAR2B interaction, inhibited Aβ25-35-induced NMDAR currents, and decreased intracellular Ca2+ levels. The effects of ST2-104 was abolished by overexpression of CRMP2 and intensified by knockdown of CRMP2 in SH-SY5Y cells. Taken together, our results support ST2-104 as a possible biologic therapeutic in the face of Aβ25-35-induced injury via the inhibition of the pCRMP2/NMDAR2B signaling pathway.

Mitochondrial morphology and motility (mitochondrial dynamics) play a major role in the proper functioning of distant synapses. In Huntington’s disease (HD), mitochondria become fragmented and less motile, but the mechanisms leading to these changes are not clear. Here, we found that collapsin response mediator protein 2 (CRMP2) interacted with Drp1 and Miro 2, proteins involved in regulating mitochondrial dynamics. CRMP2 interaction with these proteins inversely correlated with CRMP2 phosphorylation. CRMP2 was hyperphosphorylated in postmortem brain tissues of HD patients, in human neurons derived from induced pluripotent stem cells from HD patients, and in cultured striatal neurons from HD mouse model YAC128. At the same time, CRMP2 interaction with Drp1 and Miro 2 was diminished in HD neurons. The CRMP2 hyperphosphorylation and dissociation from Drp1 and Miro 2 correlated with increased fission and suppressed motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Thr 509/514 and Ser 522 and rescued CRMP2’s interaction with Drp1 and Miro 2. This was accompanied by reduced mitochondrial fission and enhanced mitochondrial motility. Additionally, (S)-LCM exerted a neuroprotective effect in YAC128 cultured neurons. Thus, our data suggest that CRMP2 may regulate mitochondrial dynamics in a phosphorylation-dependent manner and modulate neuronal survival in HD.

As neurons establish extensive connections throughout the central nervous system, the transport of cargo along the microtubule network of the axon is crucial for differentiation and homeostasis. Specifically, building blocks such as membrane and cytoskeletal components, organelles, transmembrane receptors, adhesion molecules, and peptide neurotransmitters all require proper transport to the presynaptic compartment. Here, we identify a novel complex regulating vesicular endoplasmic reticulum transport in neurites, composed of CLN6: an ER-associated protein of relatively unknown function implicated in CLN6-Batten disease; CRMP2: a tubulin binding protein important in regulating neurite microtubule dynamics; and KLC4: a classic transport motor protein. We show that this “CCK” complex allows ER-derived vesicles to migrate to the distal end of the axon, aiding in proper neurite outgrowth and arborization. In the absence of CLN6, the CCK complex does not function effectively, leading to reduced vesicular transport, stunted neurite outgrowth, and deficits in CRMP2 binding to other protein partners. Treatment with a CRMP2 modulating compound, lanthionine ketimine ester, partially restores these deficits in CLN6-deficient mouse neurons, indicating that stabilization of CRMP2 interacting partners may prove beneficial in lieu of complete restoration of the CCK complex. Taken together, these findings reveal a novel mechanism of ER-derived vesicle transport in the axon and provide new insights into therapeutic targets for neurodegenerative disease.

CRMP2/DPYL2 is an effector protein in the semaphorin signaling pathway that controls cytoskeletal dynamics, linking extracellular signals to the formation of axonal networks. CRMP2 is regulated by post-translational modifications including a dithiol-disulfide redox switch. The mechanisms of reduction of this switch were established, the signal-induced oxidation, however, remained unclear. Here, we show that CRMP2 is oxidized through a redox relay involving the flavin-mooxygenase MICAL1 and the peroxidase Prx1 as specific signal transducers. Using molecular oxygen and electrons provided by NADPH, MICAL produces hydrogen peroxide and specifically oxidizes Prx1 through direct interactions between the proteins. Subsequently, Prx1 oxidizes CRMP2. The lack of any components of this redox relay dysregulates neurite outgrowth. Consequently, both oxidation and reduction of CRMP2 require reducing equivalents in the form of NADPH.

Significance There are no biomarkers for schizophrenia (SCZ), a disorder of dysfunctional neural networks. We demonstrate that a master regulator of cytoskeleton (“CRMP2”) and, hence, neural circuitry, may form the basis for such a biomarker because its activity is uniquely imbalanced in SCZ patients. We show that SCZ patients are characterized by an excess of active CRMP2 not only in their brains (where it is correlated with dendritic abnormalities) but also in their peripheral blood lymphocytes. The abundance of active CRMP2 and insufficiency of opposing inactive p-CRMP2 likely disrupts neuronal function. Because peripheral blood CRMP2 appears to reflect intracerebral processes, it could form the basis of a rapid, minimally invasive, sensitive, and specific clinical diagnostic aid for SCZ in young patients., There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2’s activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients

Somatostatin is involved in the regulation of multiple signaling pathways and affords neuroprotection in response to neurotoxins. In the present study, we investigated the role of Somatostatin-14 (SST) in cell viability and the regulation of phosphorylation of Collapsin Response Mediator Protein 2 (CRMP2) (Ser522) via the blockade of Ca2+ accumulation, along with the inhibition of cyclin-dependent kinase 5 (CDK5) and Calpain activation in differentiated SH-SY5Y cells. Cell Viability and Caspase 3/7 assays suggest that the presence of SST ameliorates mitochondrial stability and cell survival pathways while augmenting pro-apoptotic pathways activated by A&beta, SST inhibits the phosphorylation of CRMP2 at Ser522 site, which is primarily activated by CDK5. Furthermore, SST effectively regulates Ca2+ influx in the presence of A&beta, directly affecting the activity of calpain in differentiated SH-SY5Y cells. We also demonstrated that SSTR2 mediates the protective effects of SST. In conclusion, our results highlight the regulatory role of SST in intracellular Ca2+ homeostasis. The neuroprotective role of SST via axonal regeneration and synaptic integrity is corroborated by regulating changes in CRMP2, however, SST-mediated changes in the blockade of Ca2+ influx, calpain expression, and toxicity did not correlate with CDK5 expression and p35/25 accumulation. To summarize, our findings suggest two independent mechanisms by which SST mediates neuroprotection and confirms the therapeutic implications of SST in AD as well as in other neurodegenerative diseases where the effective regulation of calcium homeostasis is required for a better prognosis.

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). If not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male and female CRMP2K374A/K374A mice, increased pain sensitivity in allodynia-resistant animals. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Effective treatment of high-impact pain patients is one of the major stated goals of the National Pain Strategy in the United States. Identification of new targets and mechanisms underlying neuropathic pain will be critical in developing new target-specific medications for better neuropathic pain management. We recently discovered that peripheral nerve injury-induced upregulation of an axonal guidance phosphoprotein collapsin response mediator protein 2 (CRMP2) and the N-type voltage-gated calcium (CaV2.2) as well as the NaV1.7 voltage-gated sodium channel, correlates with the development of neuropathic pain. In our previous studies, we found that interfering with the phosphorylation status of CRMP2 is sufficient to confer protection from chronic pain. Here we examined the expression of CRMP2 and CRMP2 phosphorylated by cyclin-dependent kinase 5 (Cdk5, on serine residue 522 (S522)) in sciatic nerve, nerve terminals of the glabrous skin, and in select subpopulations of DRG neurons in the SNI model of neuropathic pain. By enhancing our understanding of the phosphoregulatory status of CRMP2 within DRG subpopulations, we may be in a better position to design novel pharmacological interventions for chronic pain.

Collapsin response mediator proteins (CRMPs), which consist of five homologous cytosolic proteins, are one of the major phosphoproteins in the developing nervous system. The prominent feature of the CRMP family proteins is a new class of microtubule-associated proteins that play important roles in the whole process of developing the nervous system, such as axon guidance, synapse maturation, cell migration, and even in adult brain function. The CRMP C-terminal region is subjected to posttranslational modifications such as phosphorylation, which, in turn, regulates the interaction between the CRMPs and various kinds of proteins including receptors, ion channels, cytoskeletal proteins, and motor proteins. The gene-knockout of the CRMP family proteins produces different phenotypes, thereby showing distinct roles of all CRMP family proteins. Also, the phenotypic analysis of a non-phosphorylated form of CRMP2-knockin mouse model, and studies of pharmacological responses to CRMP-related drugs suggest that the phosphorylation/dephosphorylation process plays a pivotal role in pathophysiology in neuronal development, regeneration, and neurodegenerative disorders, thus showing CRMPs as promising target molecules for therapeutic intervention.

Amongst the regulators of voltage-gated ion channels is the collapsin response mediator protein 2 (CRMP2). CRMP2 regulation of the activity and trafficking of NaV1.7 voltage-gated sodium channels as well as the N-type (CaV2.2) voltage-gated calcium channel (VGCC) has been reported. On the other hand, CRMP2 does not appear to regulate L- (CaV1.x), P/Q- (CaV2.1), and R- (CaV2.3) type high VGCCs. Whether CRMP2 regulates low VGCCs remains an open question. Here, we asked if CRMP2 could regulate the low voltage-gated (T-type/CaV3.x) channels in sensory neurons. Reducing CRMP2 protein levels with short interfering RNAs yielded no change in macroscopic currents carried by T-type channels. No change in biophysical properties of the T-type currents was noted. Future studies pursuing CRMP2 druggability in neuropathic pain will benefit from the findings that CRMP2 regulates only the N-type (CaV2.2) calcium channels.

The sodium channel NaV1.7 is a master regulator of nociceptive neuronal firing. Mutations in this channel can result in painful conditions as well as produce insensitivity to pain. Despite being recognized as a “poster child” for nociceptive signaling and human pain, targeting NaV1.7 has not yet produced a clinical drug. Recent work has illuminated the NaV1.7 interactome, offering insights into the regulation of these channels and identifying potentially new druggable targets. Amongst the regulators of NaV1.7 is the cytosolic collapsin response mediator protein 2 (CRMP2). CRMP2, modified at Lysine 374 (K374) by addition of a small ubiquitin-like modifier (SUMO), bound NaV1.7 to regulate its membrane localization and function. Corollary to this, preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in rats with neuropathic pain. Notably, loss of CRMP2 SUMOylation did not compromise other innate functions of CRMP2. To further elucidate thein vivorole of CRMP2 SUMOylation in pain, we generated CRMP2 K374A knock-in (CRMP2K374A/K374A) mice in which Lys374 was replaced with Ala. CRMP2K374A/K374Amice had reduced NaV1.7 membrane localization and function in female, but not male, sensory neurons. Behavioral appraisal of CRMP2K374A/K374Amice demonstrated no changes in depressive or repetitive, compulsive-like behaviors, and a decrease in noxious thermal sensitivity. No changes were observed in CRMP2K374A/K374Amice to inflammatory, acute, or visceral pain. In contrast, in a neuropathic model, CRMP2K374A/K374Amice failed to develop persistent mechanical allodynia. Our study suggests that CRMP2 SUMOylation-dependent control of peripheral NaV1.7 is a hallmark of chronic, but not physiological, neuropathic pain.

The collapsin response mediator protein 2 (CRMP2) has emerged as a central node in assembling nociceptive signaling complexes involving voltage-gated ion channels. Concerted actions of post-translational modifications, phosphorylation and SUMOylation, of CRMP2 contribute to regulation of pathological pain states. In the present study, we demonstrate a novel role for CRMP2 in spinal nociceptive transmission. We found that, of six possible post-translational modifications, three phosphorylation sites on CRMP2 were critical for regulating calcium influx in dorsal root ganglion sensory neurons. Of these, only CRMP2 phosphorylated at serine 522 by cyclin-dependent kinase 5 (Cdk5) contributed to spinal neurotransmission in a bidirectional manner. Accordingly, expression of a non-phosphorylatable CRMP2 (S522A) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), whereas expression of a constitutively phosphorylated CRMP2 (S522D) increased the frequency of sEPSCs. The presynaptic nature of CRMP2's actions was further confirmed by pharmacological antagonism of Cdk5-mediated CRMP2 phosphorylation with S-N-benzy-2-acetamido-3-methoxypropionamide ((S)-lacosamide; (S)-LCM) which (i) decreased sEPSC frequency, (ii) increased paired-pulse ratio, and (iii) reduced the presynaptic distribution of CaV2.2 and NaV1.7, two voltage-gated ion channels implicated in nociceptive signaling. (S)-LCM also inhibited depolarization-evoked release of the pro-nociceptive neurotransmitter calcitonin gene-related peptide (CGRP) in the spinal cord. Increased CRMP2 phosphorylation in rats with spared nerve injury (SNI) was decreased by intrathecal administration of (S)-LCM resulting in a loss of presynaptic localization of CaV2.2 and NaV1.7. Together, these findings indicate that CRMP2 regulates presynaptic excitatory neurotransmission in spinal cord and may play an important role in regulating pathological pain. Novel targeting strategies to inhibit CRMP2 phosphorylation by Cdk5 may have great potential for the treatment of chronic pain.

Background and aim The aging-dependent activation of glycogen synthase kinase-3β (GSK-3β) has been suggested to be important in the onset of dementia. To discover novel therapeutic Kampo medicines for dementia, we examined the effects of orengedokuto (OGT; 黃連解毒湯 huáng lián jiědú tāng) and san'oshashinto (SST; 三黃瀉心湯 sān huáng xiè xīn tāng) on memory deficits and GSK-3β activity in senescence-accelerated prone mice (SAMP8). Experimental procedure The object recognition test (ORT) and conditioned fear memory test (CFT) were employed to elucidate short-term working memory and long-term fear memory. The activity of GSK-3β and the phosphorylation of related molecules were measured using a kinase assay and Western blotting. Results and conclusion OGT and SST attenuated memory deficits in SAMP8 in ORT, but not in CFT. In ex vivo experiments, cortical GSK-3β activity was significantly stronger in SAMP8 than in SAMR1. The enhanced cortical GSK-3β activity in SAMP8 was accompanied by a significant increase in the level of phosphorylated collapsin response mediator protein-2 (CRMP2), an important factor that is involved in the regulation of microtubule stability. OGT and SST attenuated not only increases in cortical GSK-3β activity, but also the levels of phosphorylated CRMP2 in SAMP8. In vitro experiments, flavonoids contained in these kampo medicines, inhibited GSK-3β activity in concentration-dependent manners. These results suggest that OGT and SST prevent aging-induced short-term working memory deficits by inhibiting aging-dependent elevations in the cortical GSK-3β activity and subsequent CRMP2 phosphorylation., Graphical abstract Image 1, Highlights • OGT and SST attenuated short-term working memory deficits in SAMP8. • Age-dependent cortical GSK-3β activation was suppressed by OGT and SST. • OGT and SST also attenuated the levels of phosphorylated CRMP2 in SAMP8.

Abnormal nerve regeneration often follows corneal injury, predisposing patients to pain, dry eye and vision loss. Yet, we lack a mechanistic understanding of this process. A key event in corneal wounds is the differentiation of keratocytes into fibroblasts and scar-forming myofibroblasts. Here, we show for the first time that regenerating nerves avoid corneal regions populated by myofibroblasts in vivo. Recreating this interaction in vitro, we find neurite outgrowth delayed when myofibroblasts but not fibroblasts, are co-cultured with sensory neurons. After neurites elongated sufficiently, contact inhibition was observed with myofibroblasts, but not fibroblasts. Reduced neurite outgrowth in vitro appeared mediated by transforming growth factor beta 1 (TGF-β1) secreted by myofibroblasts, which increased phosphorylation of collapsin response mediating protein 2 (CRMP2) in neurons. The significance of this mechanism was further tested by applying Mitomycin C after photorefractive keratectomy to decrease myofibroblast differentiation. This generated earlier repopulation of the ablation zone by intra-epithelial and sub-basal nerves. Our findings suggest that attaining proper, rapid corneal nerve regeneration after injury may require blocking myofibroblast differentiation and/or TGF-β during wound healing. They also highlight hitherto undefined myofibroblast-neuron signaling processes capable of restricting neurite outgrowth in the cornea and other tissues where scars and nerves co-exist.

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2’s SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

The tuber of the konjac plant is a source enriched with GlcCer (kGlcCer), and has been used as a dietary supplement to improve the dry skin and itching that are caused by a deficiency of epidermal ceramide. Previously, we showed chemoenzymatically prepared konjac ceramide has a neurite-outgrowth inhibitory effect that is very similar to that of Sema3A and is not seen with animal-type ceramides. While, it has been unclear whether kCer may act on Sema3A or TrkA signaling pathway. In the present study, we showed kCer induces phosphorylation of CRMP2 and microtubules depolymerization via Sema3A signaling pathway not TrkA. It is concluded that kCer may be a potential Sema3A-like agonist that activates Sema3A signaling pathway directly.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

While swimming exercise has been shown to positively affect the development of the nervous system, it still remains unclear whether it reduces the vulnerability to stress. In this study, male C57BL/6 mice were exposed to swimming training for 5 weeks, and then subjected to chronic unpredictable mild stress (CUMS) for 4 weeks. We found that swimming exercise prevented anxiety-like and depressive phenotypes induced by CUMS, including increased anxiety-like behavior in the open field test (OFT) and elevated plus-maze (EPM) test and increased despair behavior in the tail suspension test (TST). Moreover, the control+stress group showed reduced expression of phosphorylated AKT kinase (p-AKT), phosphorylated glycogen synthase kinase-3β (p-GSK3β), and tubulin-tyrosine ligase (Tyr-tubulin) and increased protein expression of phosphorylated collapsin response mediator protein 2 (p-CRMP-2); the control+control, swim+control, and swim+stress groups exhibited higher expression of these proteins than the control+stress group. This study confirmed that swimming exercise could reduce the vulnerability of individuals to stress and that it contributes to the AKT/GSK-3β/CRMP-2 pathway and microtubule dynamics mediated protective effects on neuroplasticity. The AKT/GSK-3β/CRMP-2 pathway and microtubule dynamics may be involved in resilience to stress.

Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.

Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood. Integrin-linked kinase (ILK) is a scaffold protein that regulates microtubule stability and F-actin dynamics. Here we show that ILK is necessary for normal trafficking of melanosomes along microtubule tracks. In the absence of ILK, immature melanosomes are not retained in perinuclear regions, and mature melanosome trafficking along microtubule tracks is impaired. These deficits can be attenuated by microtubule stabilization. Microtubules are also necessary for the formation of dendrites in melanocytes, and Ilk inactivation reduces melanocyte dendricity. Activation of glycogen synthase kinase-3 (GSK-3) interferes with microtubule assembly. Significantly, inhibition of GSK-3 activity or exogenous expression of the GSK-3 substrate collapsin response mediator protein 2 (CRMP2) in ILK-deficient melanocytes restored dendricity. ILK is also required for normal melanin transfer, and GSK-3 inhibition in melanocytes partially restored melanin transfer to neighboring keratinocytes. Thus, our work shows that ILK is a central modulator of melanosome movements in primary epidermal melanocytes and identifies ILK and GSK-3 as important modulators of melanin transfer to keratinocytes, a key process for epidermal UV photoprotection.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium’s suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium’s attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Following injury to the peripheral and central nervous systems, tissue levels of transforming growth factor (TGF)-β1 often increase, which is key for wound healing and scarring. However, active wound regions and scars appear to inhibit process outgrowth by regenerating neurons. We recently showed that corneal wound myofibroblasts block corneal nerve regeneration in vivo, and sensory neurite outgrowth in vitro in a manner that relies critically on TGF-β1. In turn, delayed, abnormal re-innervation contributes to long-term sensory dysfunctions of the ocular surface. Here, we exposed morphologically and biochemically-differentiated sensory neurons from the ND7/23 cell line to TGF-β1 to identify the intracellular signals regulating these anti-neuritogenic effects, contrasting them with those of Semaphorin(Sema)3A, a known inhibitor of neurite outgrowth. Neuronal morphology was quantified using phase-contrast imaging. Western blotting and specific inhibitors were then used to identify key molecular mediators. Differentiated ND7/23 cells expressed neuron-specific markers, including those involved in neurite extension and polarization. TGF-β1 increased phosphorylation of collapsin response mediator protein-2 (CRMP2), a molecule that is key for neurite extension. We now show that both glycogen synthase kinase (GSK)-3β and Smad3 modulate phosphorylation of CRMP2 after treatment with TGF-β1. GSK-3β appeared to exert a particularly strong effect, which could be explained by its ability to phosphorylate not only CRMP2, but also Smad3. In conclusion, TGF-β1’s inhibition of neurite outgrowth in sensory neurons appears to be regulated through a highly-conserved signaling pathway, which involves the GSK-3β/CRMP-2 loop via both canonical and non-canonical mechanisms. It is hoped that by defining the signaling pathways that control neurite outgrowth in wound environments, it will become possible to identify optimal molecular targets to promote re-innervation following injury.

Sirtuin 6 (SIRT6) has been reported to play a key role in cognitive function and mood regulation, yet its role in mood disorders is not completely understood. Here, we confirmed that knockdown of hippocampal SIRT6 alleviated depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. Our in vitro data showed that SIRT6 negatively regulated protein kinase B (AKT) signaling by deacetylating histone 3 at Lys9 and Lys56. Knockdown of SIRT6 significantly increased AKT phosphorylation activity, while decreased collapsin response mediator protein 2 (CRMP2) phosphorylation activity. Furthermore, pharmacologic inhibition of SIRT6 by ferulic acid (FA) (40 or 80 mg· kg(−1) per day, i.g.) could activate AKT/CRMP2 pathway in vitro, which has been proved to exert an antidepressant-like effect on CUS-induced depressive models. In conclusion, our study suggested that hippocampal SIRT6 contributes to the performance of depression-like behaviors by suppressing AKT/CRMP2 signaling, and FA ameliorates CUS-induced depression-like behaviors in mice as a potential pharmacologic inhibitor of SIRT6.

We previously showed that treatment with lanthionine ketimine ethyl ester (LKE) reduced disease severity and axonal damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis and increased neuronal maturation and survival in vitro. A major target of LKE is collapsin response mediator protein 2 (CRMP2), suggesting this protein may mediate LKE actions. We now show that conditional knockout of CRMP2 from neurons using a CamK2a promoter to drive Cre recombinase expression reduces disease severity in the myelin oligodendrocyte glycoprotein (MOG)35–55 EAE model, associated with decreased spinal cord axonal damage, and less glial activation in the cerebellum, but not the spinal cord. Immunohistochemical staining and quantitative polymerase chain reaction show CRMP2 depletion from descending motor neurons in the motor cortex, but not from spinal cord neurons, suggesting that the benefits of CRMP2 depletion on EAE may stem from effects on upper motor neurons. In addition, mice in which CRMP2 S522 phosphorylation was prevented by substitution for an alanine residue also showed reduced EAE severity. These results show that modification of CRMP2 expression and phosphorylation can influence the course of EAE and suggests that treatment with CRMP2 modulators such as LKE act in part by reducing CRMP2 S522 phosphorylation.

Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.

O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function. © 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

The accumulation of phosphorylated tau protein (pTau) in the entorhinal cortex (EC) is the earliest tau pathology in Alzheimer’s disease (AD). Tau tubulin kinase-1 (TTBK1) is a neuron-specific tau kinase and expressed in the EC and hippocampal regions in both human and mouse brains. Here we report that collapsin response mediator protein-2 (CRMP2), a critical mediator of growth cone collapse, is a new downstream target of TTBK1 and is accumulated in the EC region of early stage AD brains. TTBK1 transgenic mice show severe axonal degeneration in the perforant path, which is exacerbated by crossing with Tg2576 mice expressing Swedish familial AD mutant of amyloid precursor protein (APP). TTBK1 mice show accumulation of phosphorylated CRMP2 (pCRMP2), in the EC at 10 months of age, whereas age-matched APP/TTBK1 bigenic mice show pCRMP2 accumulation in both the EC and hippocampal regions. Amyloid-β peptide (Aβ) and TTBK1 suppresses the kinetics of microtubule polymerization and TTBK1 reduces the neurite length of primary cultured neurons in Rho kinase-dependent manner in vitro. Silencing of TTBK1 or expression of dominant-negative Rho kinase demonstrates that Aβ induces CRMP2 phosphorylation at threonine 514 in a TTBK1-dependent manner, and TTBK1 enhances Aβ-induced CRMP2 phosphorylation in Rho kinase-dependent manner in vitro. Furthermore, TTBK1 expression induces pCRMP2 complex formation with pTau in vitro, which is enhanced upon Aβ stimulation in vitro. Finally, pCRMP2 forms a complex with pTau in the EC tissue of TTBK1 mice in vivo, which is exacerbated in both the EC and hippocampal tissues in APP/TTBK1 mice. These results suggest that TTBK1 and Aβ synergistically induce phosphorylation of CRMP2, which may be causative for the neurite degeneration and somal accumulation of pTau in the EC neurons, indicating critical involvement of TTBK1 and pCRMP2 in the early AD pathology.

The dynamic trafficking of AMPA receptors (AMPARs), which enables the endocytosis, recycling, and exocytosis of AMPARs, is crucial for synaptic plasticity. Endophilin2, which directly interacts with the GluA1 subunit of AMPARs, plays an important role in AMPAR endocytosis. Collapsin response mediator protein 2 (CRMP2) promotes the maturation of the dendritic spine and can transfer AMPARs to the membrane. Although the mechanisms of AMPAR endocytosis and exocytosis are well known, the exact molecular mechanisms underlying AMPAR recycling remain unclear. Here, we report a unique interaction between CRMP2 and endophilin2. Our results showed that overexpression of CRMP2 and endophilin2 increased the amplitude and frequency of miniature excitatory synaptic currents (mEPSCs) and modestly enhanced AMPAR levels in hippocampal neurons. Furthermore, the CRMP2 and endophilin2 overexpression phenotype failed to occur when the interaction between these two proteins was inhibited. Further analysis revealed that this interaction was regulated by CRMP2 phosphorylation. The phosphorylation of CRMP2 inhibited its interaction with endophilin2; this was mainly affected by the phosphorylation of Thr514 and Ser518 by glycogen synthase kinase (GSK) 3β. CRMP2 phosphorylation increased degradation and inhibited the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. However, the dephosphorylation of CRMP2 inhibited degradation and promoted the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. Taken together, our data demonstrated that the interaction between CRMP2 and endophilin2 was conductive to the recycling of AMPA receptor GluA1 subunits in hippocampal neurons.

Biomarkers and effective therapeutic agents to improve the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) are urgently required. We aimed to analyze the prognostic value and mechanistic action of miR-93 in PDAC. Correlation of miR-93 tumor levels from 83 PDAC patients and overall survival (OS) was analyzed by Kaplan–Meier. MiR-93 depletion in PANC-1 and MIA PaCa-2 cells was achieved by CRISPR/Cas9 and miR-93 overexpression in HPDE cells by retroviral transduction. Cell proliferation, migration and invasion, cell cycle analysis, and in vivo tumor xenografts in nude mice were assessed. Proteomic analysis by mass spectrometry and western-blot was also performed. Finally, miR-93 direct binding to candidate mRNA targets was evaluated by luciferase reporter assays. High miR-93 tumor levels are significantly correlated with a worst prognosis in PDAC patients. MiR-93 abolition altered pancreatic cancer cells phenotype inducing a significant increase in cell size and a significant decrease in cell invasion and proliferation accompanied by a G2/M arrest. In vivo, lack of miR-93 significantly impaired xenograft tumor growth. Conversely, miR-93 overexpression induced a pro-tumorigenic behavior by significantly increasing cell proliferation, migration, and invasion. Proteomic analysis unveiled a large group of deregulated proteins, mainly related to G2/M phase, microtubule dynamics, and cytoskeletal remodeling. CRMP2, MAPRE1, and YES1 were confirmed as direct targets of miR-93. MiR-93 exerts oncogenic functions by targeting multiple genes involved in microtubule dynamics at different levels, thus affecting the normal cell division rate. MiR-93 or its direct targets (CRMP2, MAPRE1, or YES1) are new potential therapeutic targets for PDAC.