Anti-CRMP2 antibody induces anxiety-like behavior and increases pyramidal neuron excitability in mice.

BACKGROUND: We have previously identified auto-antibody (Ab) to collapsin response mediator protein 2 (CRMP2) in patients with encephalitis. The present study aims to evaluate the pathogenic effects of anti-CRMP2 Ab. METHODS: Recombinant CRMP2 protein was injected subcutaneously into mice to establish an active immune mouse model with anti-CRMP2 Ab. Behavioral assessments, histopathological staining, and electrophysiological testing were performed to identify any pathogenic changes. RESULTS: The mice exhibited signs of impaired motor coordination four weeks post-immunization of CRMP2 protein. Moreover, CRMP2 immunized mice for eight weeks showed anxiety-like behaviors indicating by tests of open field and the elevated plus maze. After incubating the CA1 region of hippocampal brain section with the sera from CRMP2 immunized mice, the whole-cell path-clamp recordings showed increased excitability of pyramidal neurons. However, no obvious inflammation and infiltration of immune cells were observed by histopathological analysis. Western blot showed that the phosphorylation levels of CRMP2-Thr514 and -Ser522 were not affected. CONCLUSION: In an active immunization model with CRMP2 protein, impaired coordination and anxiety-like behaviors were observed. Also, anti-CRMP2 Abs containing sera heightened the excitability of hippocampal pyramidal neurons in vitro, which imply the pathogenic effects of anti-CRMP2 Ab.

Clinical characteristics and genetic analysis of children with Omicron BF.7.14 type novel coronavirus-related acute necrotizing encephalopathy.

Background: To explore the clinical characteristics, etiological factors, and clinical-related genetic variant of children with acute necrotizing encephalopathy (ANE) related to the Omicron BF.7.14 novel coronavirus. Methods: Genomic variations were detected through whole exome sequencing. Additionally, we summarized the clinical data to explore the inheritance patterns associated with novel coronavirus-related ANE. Results: This study included four patients (2 males and 2 females) with an average age of 2.78 ± 1.93 years. All the patients had prodromal symptoms of Omicron BF.7.14 virus infection, and exhibited symptoms such as altered consciousness, seizures and cognitive/language disturbances. Cranial MRI scans revealed damage to the thalamus, basal ganglia and brainstem. The cerebrospinal fluid (CSF) cell counts were nearly normal, but protein level in CSF increased significantly. Genetic analysis revealed a novel truncated variant of CRMP2 gene in one patient who suffered more severe coma score and prognosis and dead in the later stages. All children exhibited a decrease in the absolute count of T lymphocytes, helper T cells, suppressor T cells, and NK cells to varying degrees. Furthermore, levels of cytokines, including IL-1 ß, IL-5, IL-6 and IL-8 were significantly elevated in the CSF, especially in patient with truncated variant of CRMP2 gene. Conclusion: The Omicron BF.7.14 type novel coronavirus can lead to ANE, characterized by T cell immunosuppression and a significant increase in cytokine levels in the CSF. The truncated variation of CRMP2 gene may affect the prognosis of ANE by affecting the migration of cerebral T cells.

Inhibition of ERK1/2 or CRMP2 Disrupts Alcohol Memory Reconsolidation and Prevents Relapse in Rats.

Relapse to alcohol abuse, often caused by cue-induced alcohol craving, is a major challenge in alcohol addiction treatment. Therefore, disrupting the cue-alcohol memories can suppress relapse. Upon retrieval, memories transiently destabilize before they reconsolidate in a process that requires protein synthesis. Evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), governing the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase 1/2 (ERK1/2), in the reconsolidation process in a rat (Wistar) model of alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, while the PI3K-AKT pathway remained unaffected. Importantly, ERK1/2 inhibition after alcohol memory retrieval impaired alcohol-memory reconsolidation and led to long-lasting relapse suppression. Attenuation of relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2)-a translational product of mTORC1. Together, our findings indicate the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, with their inhibition as potential treatment targets for relapse prevention.

Drug Treatment Attenuates Retinal Ganglion Cell Death by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation in Mouse Models of Normal Tension Glaucoma.

Normal tension glaucoma (NTG) is a progressive neurodegenerative disease in glaucoma families. Typical glaucoma develops because of increased intraocular pressure (IOP), whereas NTG develops despite normal IOP. As a subtype of open-angle glaucoma, NTG is characterized by retinal ganglion cell (RGC) degeneration, gradual loss of axons, and injury to the optic nerve. The relationship between glutamate excitotoxicity and oxidative stress has elicited great interest in NTG studies. We recently reported that suppressing collapsin response mediator protein 2 (CRMP2) phosphorylation in S522A CRMP2 mutant (CRMP2 KIKI) mice inhibited RGC death in NTG mouse models. This study evaluated the impact of the natural compounds huperzine A (HupA) and naringenin (NAR), which have therapeutic effects against glutamate excitotoxicity and oxidative stress, on inhibiting CMRP2 phosphorylation in mice intravitreally injected with N-methyl-D-aspartate (NMDA) and GLAST mutant mice. Results of the study demonstrated that HupA and NAR significantly reduced RGC degeneration and thinning of the inner retinal layer, and inhibited the elevated CRMP2 phosphorylation. These treatments protected against glutamate excitotoxicity and suppressed oxidative stress, which could provide insight into developing new effective therapeutic strategies for NTG.

Insular cortex stimulation alleviates neuropathic pain through changes in the expression of collapsin response mediator protein 2 involved in synaptic plasticity.

In recent studies, brain stimulation has shown promising potential to alleviate chronic pain. Although studies have shown that stimulation of pain-related brain regions can induce pain-relieving effects, few studies have elucidated the mechanisms of brain stimulation in the insular cortex (IC). The present study was conducted to explore the changes in characteristic molecules involved in pain modulation mechanisms and to identify the changes in synaptic plasticity after IC stimulation (ICS). Following ICS, pain-relieving behaviors and changes in proteomics were explored. Neuronal activity in the IC after ICS was observed by optical imaging. Western blotting was used to validate the proteomics data and identify the changes in the expression of glutamatergic receptors associated with synaptic plasticity. Experimental results showed that ICS effectively relieved mechanical allodynia, and proteomics identified specific changes in collapsin response mediator protein 2 (CRMP2). Neuronal activity in the neuropathic rats was significantly decreased after ICS. Neuropathic rats showed increased expression levels of phosphorylated CRMP2, alpha amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR), and N-methyl-d-aspartate receptor (NMDAR) subunit 2B (NR2B), which were inhibited by ICS. These results indicate that ICS regulates the synaptic plasticity of ICS through pCRMP2, together with AMPAR and NR2B, to induce pain relief.

Effects of esketamine on depression-like behavior and dendritic spine plasticity in the prefrontal cortex neurons of spared nerve injury-induced depressed mice

The present study utilized the spared nerve injury (SNI) to create a mouse model of depression to investigate the impact of esketamine on depressive-like behaviors, on the expression of PSD-95 and CRMP2 proteins, and on changes in neuronal dendritic spine plasticity in the prefrontal cortex (PFC). Depressive-like behavioral tests were performed 1 h after esketamine treatment, and the PFC tissues were obtained on the fourth day after completing the behavioral tests. Then, dendritic spine density and morphology in the PFC were measured using Golgi staining, and CRMP2 and PSD-95 proteins were obtained from PFC tissue by western blotting. The results of this study showed that esketamine significantly increased the immobility time in the forced swimming test and tail suspension test. In the open field test, esketamine increased the time spent in the open arms, the time spent in the central area, and the total distance covered. It also increased the protein expression levels of CRMP2 and PSD-95 in addition to the total and mature dendritic spine density of the PFC in SNI-depressed mice. Esketamine can significantly improve depression-like behaviors in SNI-depressed mice and promote an increase in dendritic spine density and maturation in the PFC. These effects may be associated with changes in CRMP2 and PSD-95 expression.

Sinomenine ameliorates fibroblast-like synoviocytes dysfunction by promoting phosphorylation and nuclear translocation of CRMP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and arthritic pain. Sinomenine (SIN), derived from the rhizome of Chinese medical herb Qing Teng (scientific name: Sinomenium acutum (Thunb.) Rehd. Et Wils), has a longstanding use in Chinese traditional medicine for treating rheumatoid arthritis. It has been shown to possess anti-inflammatory, analgesic, and immunosuppressive effects with minimal side-effects clinically. However, the mechanisms governing its effects in treatment of joint pathology, especially on fibroblast-like synoviocytes (FLSs) dysfunction, and arthritic pain remains unclear. AIM: This study aimed to investigate the effect and underlying mechanism of SIN on arthritic joint inflammation and joint FLSs dysfunctions. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) was induced in rats and the therapeutic effects of SIN on joint pathology were evaluated histopathologically. Next, we conducted a series of experiments using LPS-induced FLSs, which were divided into five groups (Naïve, LPS, SIN 10, 20, 50 µg/ml). The expression of inflammatory factors was measured by qPCR and ELISA. The invasive ability of cells was detected by modified Transwell assay and qPCR. Transwell migration and cell scratch assays were used to assess the migration ability of cells. The distribution and content of relevant proteins were observed by immunofluorescence and laser confocal microscopy, as well as Western Blot and qPCR. FLSs were transfected with plasmids (CRMP2 T514A/D) to directly modulate the post-translational modification of CRMP2 protein and downstream effects on FLSs function was monitored. RESULTS: SIN alleviated joint inflammation in rats with CIA, as evidenced by improvement of synovial hyperplasia, inflammatory cell infiltration and cartilage damage, as well as inhibition of pro-inflammatory cytokines release from FLSs induced by LPS. In vitro studies revealed a concentration-dependent suppression of SIN on the invasion and migration of FLSs induced by LPS. In addition, SIN downregulated the expression of cellular CRMP2 that was induced by LPS in FLSs, but increased its phosphorylation at residue T514. Moreover, regulation of pCRMP2 T514 by plasmids transfection (CRMP2 T514A/D) significantly influenced the migration and invasion of FLSs. Finally, SIN promoted nuclear translocation of pCRMP2 T514 in FLSs. CONCLUSIONS: SIN may exert its anti-inflammatory and analgesic effects by modulating CRMP2 T514 phosphorylation and its nuclear translocation of FLSs, inhibiting pro-inflammatory cytokine release, and suppressing abnormal invasion and migration. Phosphorylation of CRMP2 at the T514 site in FLSs may present a new therapeutic target for treating inflammatory joint's destruction and arthritic pain in RA.

Effect of lidocaine on cognitively impaired rats: Anti-inflammatory and antioxidant mechanisms in combination with CRMP2 antiphosphorylation.

OBJECTIVE: Studies have shown that lidocaine has antioxidative stress, anti-inflammatory, and nerve-protective effects. The current study investigated the effects of lidocaine on cognitive function in rats with cognitive dysfunction. METHODS: A total of 48 rats were randomly assigned to four groups of 12 rats each: control group; L (lidocaine) + D (d-galactose) group, d-galactose group (D group); and D + L group. We assessed cognitive function using a Morris water maze (MWM) and pathologic changes of hippocampal sections. An enzyme-linked immunosorbent assay (ELIZA) was used to detect serum malondialdehyde (MDA) and superoxide dismutase (SOD) levels in rats, and protein immunoblotting (western blot) was used to detect brain tissue proteins (collapsing response mediator protein-2 [CRMP2], phosphorylated-collapsing response mediator protein-2 [P-CRMP2], and ß-amyloid protein [Aß]). RESULTS: The MWM showed that the d-gal group (284.09 ± 20.46, 5.20 ± 0.793) performed worse than the L + D (265.37 ± 22.34, 4.170 ± 0.577; p = .000) and D + L groups (254.72 ± 27.87, 3.750; p = .000) in escape latency and number of platform crossings, respectively. The L + D group (44.94 ± 2.92 pg/mL, 6.22 ± 0.50 pg/mL, and 460.02 ± 8.26 nmol/mL) and D + L group (46.88 ± 2.63 pg/mL, 5.90 ± 0.38 pg/mL, and 465.6 ± 16.07 nmol/mL) had significantly lower serum inflammatory levels of interleukin-6, tumor necrosis factor-α, and MDA than the d-gal group (57.79 ± 3.96 pg/mL, 11.25 ± 1.70 pg/mL, and 564.9 ± 15.90 nmol/mL), respectively. The L + D group (3.17 ± 0.41 µg/mL) and D + L group (3.08 ± 0.09 µg/mL) had significantly higher serum inflammatory levels of SOD than the d-gal group (2.20 ± 0.13 µg/mL) (all p = .000). The levels of CRMP2, P-CRMP2, and Aß in the brain tissue homogenates of the L + D group (0.87 ± 0.04, 0.57 ± 0.0, and 0.16 ± 0.02) and the D + L group (0.82 ± 0.05, 0.58 ± 0.09, and 0.15 ± 0.02) were significantly different than the d-gal group (0.67 ± 0.03, 0.96 ± 0.040, and 0.29 ± 0.05). CONCLUSIONS: Lidocaine was shown to reduce cognitive impairment in rats with cognitive dysfunction through anti-inflammatory and antioxidative stress mechanisms in combination with CRMP2 antiphosphorylation.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

CRMP2 conditional knockout changes axonal function and ultrastructure of axons in mice corpus callosum.

Collapsin response mediator protein 2 (CRMP2) is a member of a protein family, which is highly involved in neurodevelopment, but most of its members become heavily downregulated in adulthood. CRMP2 is an important factor in neuronal polarization, axonal formation and growth cone collapse. The protein remains expressed in adulthood, but is more region specific. CRMP2 is present in adult corpus callosum (CC) and in plastic areas like prefrontal cortex and hippocampus. CRMP2 has been implicated as one of the risk-genes for Schizophrenia (SZ). Here, a CRMP2 conditional knockout (CRMP2-cKO) mouse was used as a model of SZ to investigate how it could affect the white matter and therefore brain connectivity. Multielectrode electrophysiology (MEA) was used to study the function of corpus callosum showing an increase in conduction velocity (CV) measured as Compound Action Potentials (CAPs) in acute brain slices. Light- and electron-microscopy, specifically Serial Block-face Scanning Electron Microscopy (SBF-SEM), methods were used to study the structure of CC in CRMP2-cKO mice. A decrease in CC volume of CRMP2-cKO mice as compared to controls was observed. No differences were found in numbers nor in the size of CC oligodendrocytes (OLs). Similarly, no differences were found in myelin thickness or in node of Ranvier (NR) structure. In contrast, abnormally smaller axons were measured in the CRMP2-cKO mice. Using these state-of-the-art methods it was possible to shed light on specific parts of the dysconnectivity aspect of deletion of CRMP2 related to SZ and add details to previous findings helping further understanding the disease. This paper substantiates the white matter changes in the absence of CRMP2 and ties it to the role it plays in this complex disorder.

CRMP2 Participates in Regulating Mitochondrial Morphology and Motility in Alzheimer's Disease.

Mitochondrial bioenergetics and dynamics (alterations in morphology and motility of mitochondria) play critical roles in neuronal reactions to varying energy requirements in health and disease. In Alzheimer's disease (AD), mitochondria undergo excessive fission and become less motile. The mechanisms leading to these alterations are not completely clear. Here, we show that collapsin response mediator protein 2 (CRMP2) is hyperphosphorylated in AD and that is accompanied by a decreased interaction of CRMP2 with Drp1, Miro 2, and Mitofusin 2, which are proteins involved in regulating mitochondrial morphology and motility. CRMP2 was hyperphosphorylated in postmortem brain tissues of AD patients, in brain lysates, and in cultured cortical neurons from the double transgenic APP/PS1 mice, an AD mouse model. CRMP2 hyperphosphorylation and dissociation from its binding partners correlated with increased Drp1 recruitment to mitochondria, augmented mitochondrial fragmentation, and reduced mitochondrial motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Ser 522 and Thr 509/514, and restored CRMP2's interaction with Miro 2, Drp1, and Mitofusin 2. This was paralleled by decreased Drp1 recruitment to mitochondria, diminished mitochondrial fragmentation, and improved motility of the organelles. Additionally, (S)-LCM-protected cultured cortical AD neurons from cell death. Thus, our data suggest that CRMP2, in a phosphorylation-dependent manner, participates in the regulation of mitochondrial morphology and motility, and modulates neuronal survival in AD.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3ß, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

Cell specific regulation of NaV1.7 activity and trafficking in rat nodose ganglia neurons.

The voltage-gated sodium NaV1.7 channel sets the threshold for electrogenesis. Mutations in the gene encoding human NaV1.7 (SCN9A) cause painful neuropathies or pain insensitivity. In dorsal root ganglion (DRG) neurons, activity and trafficking of NaV1.7 are regulated by the auxiliary collapsin response mediator protein 2 (CRMP2). Specifically, preventing addition of a small ubiquitin-like modifier (SUMO), by the E2 SUMO-conjugating enzyme Ubc9, at lysine-374 (K374) of CRMP2 reduces NaV1.7 channel trafficking and activity. We previously identified a small molecule, designated 194, that prevented CRMP2 SUMOylation by Ubc9 to reduce NaV1.7 surface expression and currents, leading to a reduction in spinal nociceptive transmission, and culminating in normalization of mechanical allodynia in models of neuropathic pain. In this study, we investigated whether NaV1.7 control via CRMP2-SUMOylation is conserved in nodose ganglion (NG) neurons. This study was motivated by our desire to develop 194 as a safe, non-opioid substitute for persistent pain, which led us to wonder how 194 would impact NaV1.7 in NG neurons, which are responsible for driving the cough reflex. We found functioning NaV1.7 channels in NG neurons; however, they were resistant to downregulation via either CRMP2 knockdown or pharmacological inhibition of CRMP2 SUMOylation by 194. CRMP2 SUMOylation and interaction with NaV1.7 was consered in NG neurons but the endocytic machinery was deficient in the endocytic adaptor protein Numb. Overexpression of Numb rescued CRMP2-dependent regulation on NaV1.7, rendering NG neurons sensitive to 194. Altogether, these data point at the existence of cell-specific mechanisms regulating NaV1.7 trafficking.

Neuronal CRMP2 phosphorylation inhibition by the flavonoid, naringenin, contributes to the reversal of spinal sensitization and arthritic pain improvement.

BACKGROUND: Rheumatoid arthritis patients usually suffer from arthritic chronic pain. However, due to an incomplete understanding of the mechanisms underlying autoimmune disorders, the management of arthritic pain is unsatisfactory. Here, we investigated the analgesic effect and underlying mechanism of the natural flavonoid naringenin (NAR) in collagen-induced arthritis (CIA) pain. METHODS: NAR was injected (i.p.) once per day for 42 days after initial immunization, and rats were sacrificed on the 28th (the 21st day after final immunization, PID 21) and 42nd days (PID 35). The inflammatory factors, central sensitization indicators, and CRMP2 phosphorylation, as well as the anti-rheumatoid activity and analgesic effect of NAR, were further investigated. RESULTS: We found that NAR decreased the arthritis score and paw swelling, as well as the mechanical and thermal pain. The immunofluorescence results also showed a dose dependent effect of NAR on reducing the expressions of spinal cFos, IBA-1, and GFAP on the 28th (PID 21) and 42nd day (PID 35). NAR decreased the phosphorylation of CRMP2 S522 and the expression of the kinase CDK5 in the spinal dorsal horn, but pCRMP2 Y479 was unchanged. In addition, CRMP2 was co-localized with NEUN, but not IBA-1 or GFAP, indicating the involvement of neural CRMP2 phosphorylation in CIA-related pain. Finally, CRMP2 S522 phosphorylation selective inhibitor (S)-lacosamide also alleviated arthritic pain. CONCLUSIONS: Taken together, our results demonstrate that NAR alleviates inflammation and chronic pain in CIA model, which might be related to its inhibition of neuronal CRMP2 S522 phosphorylation, potentially mitigating the central sensitization. Our study provide evidence for the potential use of NAR as non-opioid-dependent analgesia in arthritic pain.

Cdc42 Promotes Axonogenesis of Primary Hippocampal Neurons by Inhibiting Glycogen Synthase Kinase-3ß.

BACKGROUND: Progressive axon degeneration is a common pathological feature of neurodegenerative diseases. Cdc42 is a member of the Rho GTPase family that participates in axonogenesis. GSK-3ß is a serine/threonine kinase highly implicated in neuronal development and neurodegeneration. This study aimed to examine whether cdc42 promotes axonogenesis by regulating GSK-3ß activity. METHODS: Hippocampal neurons were isolated from neonatal Sprague-Dawley rats and transfected with designated plasmid vectors to alter the activities of cdc42 and GSK-3ß. LiCl treatment was used to inhibit the GSK-3ß activity in primary neurons. GSK-3ß activity was determined by an enzyme activity assay kit. Immunofluorescence staining was used to detect axons stained with anti-Tau-1 antibody and dendrites stained with anti-MAP2 antibody. RESULTS: Transfection with an active cdc42 mutant (cdc42F28L) decreased the activity of GSK-3ß and induced axonogenesis in primary rat hippocampal neurons, while transfection with a negative cdc42 mutant (cdc42N17) resulted an opposite effect. Moreover, transfection with plasmid vectors carrying wild-type GSK-3ß or a constitutively active GSK3ß mutant (GSK-3ß S9A) increased the activity of GSK-3ß and attenuated axonogenesis of primary hippocampal neurons with excessive cdc42 activity, whereas inhibition of GSK-3ß by LiCl abolished the inhibitory effect of the negative cdc42 mutant on axonogenesis. CONCLUSIONS: This study suggests that cdc42 induces axonogenesis of primary rat hippocampal neurons via inhibiting GSK-3ß activity. These findings support further investigation into the mechanisms of cdc42/GSK-3ß-mediated axonogenesis.

CRMP2 and CRMP4 are required for the formation of commissural tracts in the developing zebrafish forebrain.

Axonal connections between the two sides of the brain are essential for processing sensorimotor functions, especially in animals with bilateral symmetry. The anterior commissure and postoptic commissure are two crucial axonal projections that develop early in the zebrafish central nervous system. In this study, we characterized the function of collapsin response mediator protein 2 (CRMP2) and CRMP4 in patterning the development of the anterior and postoptic commissures by analyzing morpholino-knockdown zebrafish morphants and CRISPR/Cas9-edited gene-knockout mutants. We observed a loss of commissural structures or a significant reduction in axon bundles connecting the two hemispheres, but the defects could be largely recovered by co-injecting CRMP2 or CRMP4 mRNA. Loss of both CRMP2 and CRMP4 function resulted in a synergistic increase in the number of commissural defects. To elucidate the mechanism by which CRMP2 and CRMP4 provide guidance cues for the development of the anterior and postoptic commissures, we included neuropilin 1a (Nrp1a) morphants and double morphants (CRMP2/Nrp1a and CRMP4/Nrp1a) for analysis. Our experimental results indicated that CRMP2 and CRMP4 might mediate their activities through the common semaphorin 3/Nrp1a signaling pathway.

Neuropilin-2 Signaling Modulates Mossy Fiber Sprouting by Regulating Axon Collateral Formation Through CRMP2 in a Rat Model of Epilepsy.

Programmed neural circuit formation constitutes the foundation for normal brain functions. Axon guidance cues play crucial roles in neural circuit establishment during development. Whether or how they contribute to maintaining the stability of networks in mature brains is seldom studied. Upon injury, neural rewiring could happen in adulthood, of which mossy fiber sprouting (MFS) is a canonical example. Here, we uncovered a novel role of axon guidance molecule family Sema3F/Npn-2 signaling in MFS and epileptogenesis in a rat model of epilepsy. Dentate gyrus-specific Npn-2 knockdown increased seizure activity in epileptic animals along with increased MFS. Hippocampal culture results suggested that Npn-2 signaling modulates MFS via regulating axon outgrowth and collateral formation. In addition, we discovered that Sema3F/Npn-2 signal through CRMP2 by regulating its phosphorylation in the process of MFS. Our work illustrated that Npn-2 signaling in adult epilepsy animals could potentially modulate seizure activity by controlling MFS. MFS constitutes the structural basis for abnormal electric discharge of neurons and recurrent seizures. Therapies targeting Npn-2 signaling could potentially have disease-modifying anti-epileptogenesis effects in epilepsy treatment.

Effects of Lanthionine Ketimine-5-Ethyl Ester on the α-Synucleinopathy Mouse Model.

Potentially druggable mechanisms underlying synaptic deficits seen in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are under intense interrogations. In addition to defective synaptic vesicle trafficking, cytoskeletal disruption, autophagic perturbation, and neuroinflammation, hyperphosphorylation of microtubule-associated protein collapsin response mediator protein 2 (CRMP2, also known as DPYSL2) is newly determined to correlate with synaptic deficits in human DLB. The small molecule experimental therapeutic, lanthionine ketimine-5-ethyl ester (LKE), appears to interact with CRMP2 in a host of neurodegenerative mouse models, normalizing its phosphorylation level while promoting healthful autophagy in cell culture models and suppressing the proinflammatory phenotype of activated microglia. Accordingly, this study examined the effect of LKE on α-synuclein A53T transgenic (Tg) mice which were employed as a DLB model. We found that chronic administration of LKE to A53T mice suppressed (1) the accumulation of LBs, (2) neuroinflammatory activation of microglia, (3) impairment of contextual fear memory, and (4) CRMP2 phosphorylation at Thr509 in A53T Tg mice. These results suggest that CRMP2 phosphorylation by GSK3ß in the hippocampus is related to pathology and memory impairment in DLB, and LKE may have clinical implications in the treatment of α-synucleinopathy.

Identification of Anti-Collapsin Response Mediator Protein 2 Antibodies in Patients With Encephalitis or Encephalomyelitis.

Background and Purpose: An increasing number of autoimmune encephalitis (AE)-associated autoantibodies have been successfully characterized. However, many cases of AE remain unexplained on account of unknown antibodies. The aim of the present study was to identify a novel antibody against collapsin response mediator protein 2 (CRMP2) in suspected AE patients. Methods: A patient's serum and cerebrospinal fluid samples tested negative for known AE antibodies; however, strong immunolabel signals were observed in the neuronal cytoplasm of the cortex, hippocampus, and Purkinje cells on rat brain sections. Immunoprecipitation from the rat brain protein lysate, followed by mass spectrometry analysis, was used to identify the targeting antigen. Western blotting and cell-based assay with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope, IgG subtype determination, and retrospective study. Results: An antibody against CRMP2, a synaptic protein involved in axon guidance, was identified. The immunostains of the patient's samples on rat brain sections were eliminated by pre-absorption with HEK293T cells overexpressing CRMP2. The samples specifically immunoreacted with CRMP2, but not with CRMP1, CRMP3, CRMP4, and CRMP5. The C-terminus of CRMP2 with 536 amino acids contained the epitope for antibody binding. The subtype analysis showed that the anti-CRMP2 antibody was IgG4. Furthermore, a screening of 46 patients with neurological disoders and neuro-cytoplasm immunostainings on rat brain sections resulted in the identification of anti-CRMP2 antibodies in a case of encephalomyelitis. The two patients responded well to immunotherapies. Conclusions: This study discovered that a novel anti-CRMP2 antibody was associated with suspected AE and thus should be included in the testing list for AE.