Collapsin Response Mediator Protein 2 Expression Research Articles

Riluzole Promotes Neurite Growth in Rats after Spinal Cord Injury through the GSK-3β/CRMP-2 Pathway.

Spinal cord injury (SCI) is a disastrous event that often leads to permanent neurological deficits involving motor, sensory, and autonomic dysfunctions in patients. Accumulating research has demonstrated that riluzole may play crucial roles in the process of spinal tissue repair, but the underlying mechanisms remain elusive. This study verified the effectiveness of riluzole and speculated that a riluzole-afforded protection mechanism may be associated with the glycogen synthase kinase-3 beta (GSK-3β)/collapsin response mediator protein-2 (CRMP-2) pathway in rats after spinal cord injury. Here, a modified Allen's weight dropping model was generated and riluzole at 4 mg/kg was injected intraperitoneally after surgery and twice a day for 7 consecutive days. At 6 weeks after SCI, we found that riluzole treatment reduced the central cavity size of the spinal cord and improved neurological functions. Meanwhile, riluzole-treated rats exhibited shorter latency and larger amplitude in motor evoked potentials and somatosensory evoked potentials, compared with vehicle-treated rats. Furthermore, Western blotting and immunofluorescence data revealed that the expression levels of GSK-3β and phosphorylated-GSK-3β were lower in riluzole-treated SCI rats compared with vehicle-treated rats. We next detected the expression CRMP-2 and phosphorylated CRMP-2 and found that the expression of CRMP-2 showed no difference between the riluzole-treated and vehicle-treated groups; however, administration of riluzole downregulated phosphorylated CRMP-2 expression. The current findings suggest that after SCI, administration of riluzole promotes neurological functional restoration, which may be associated, in part, with its activation of the GSK-3β/CRMP-2 signaling pathway.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

CRMP2 Is Involved in Regulation of Mitochondrial Morphology and Motility in Neurons.

Regulation of mitochondrial morphology and motility is critical for neurons, but the exact mechanisms are unclear. Here, we demonstrate that these mechanisms may involve collapsin response mediator protein 2 (CRMP2). CRMP2 is attached to neuronal mitochondria and binds to dynamin-related protein 1 (Drp1), Miro 2, and Kinesin 1 light chain (KLC1). Treating neurons with okadaic acid (OA), an inhibitor of phosphatases PP1 and PP2A, resulted in increased CRMP2 phosphorylation at Thr509/514, Ser522, and Thr555, and augmented Drp1 phosphorylation at Ser616. The CRMP2-binding small molecule (S)-lacosamide ((S)-LCM) prevented an OA-induced increase in CRMP2 phosphorylation at Thr509/514 and Ser522 but not at Thr555, and also failed to alleviate Drp1 phosphorylation. The increased CRMP2 phosphorylation correlated with decreased CRMP2 binding to Drp1, Miro 2, and KLC1. (S)-LCM rescued CRMP2 binding to Drp1 and Miro 2 but not to KLC1. In parallel with CRMP2 hyperphosphorylation, OA increased mitochondrial fission and suppressed mitochondrial traffic. (S)-LCM prevented OA-induced alterations in mitochondrial morphology and motility. Deletion of CRMP2 with a small interfering RNA (siRNA) resulted in increased mitochondrial fission and diminished mitochondrial traffic. Overall, our data suggest that the CRMP2 expression level and phosphorylation state are involved in regulating mitochondrial morphology and motility in neurons.

Effects of CRMP2 DNA Methylation in the Hippocampus on Depressive-Like Behaviors and Cytoskeletal Proteins in Rats.

Chronic stress appears to alter DNA methylation and DNA methyltransferases (DNMTs) in brain regions related to emotion. Collapsin response mediator protein-2 (CRMP2) mediates the development of depression by regulating microtubule dynamics. In this study, rats were subjected to chronic unpredictable mild stress (CUMS). At the end of the CUMS procedure, normal saline or fluoxetine was administered to the rats. Moreover, normal saline or the 5-aza-2’-deoxycytidine (5-aza) was administered to the hippocampal CA1 region of the rats. Behavioral tests were performed to evaluate the depressive-like phenotypes. The CRMP2 DNA methylation levels and cytoskeletal microtubular system-related biomarkers were detected by several molecular biology techniques. The results showed that the rat model of depression was successfully established by exposure to CUMS, and fluoxetine treatment exerted an antidepressant-like effect. We observed the upregulation of DNMT1 and DNMT3a in the hippocampus of stressed rats. CUMS induced a decrease in CRMP2 expression and an increase in phosphorylated CRMP2 (pCRMP2) expression in the hippocampus of rats. The rate of DNA methylation in the CpG island of the CRMP2 promoter region in the hippocampus of stressed rats was significantly higher than that in control rats. Moreover, CUMS significantly decreased the interaction between CRMP2 and α-tubulin and decreased the microtubule dynamics. Chronic fluoxetine treatment reversed these changes. Also, hypomethylation induced by 5-aza injection into the hippocampal CA1 region caused antidepressant-like effects and increased CRMP2 expression and microtubule dynamics. These results suggested that CRMP2 DNA methylation may be involved in regulating the cytoskeletal microtubular system and mediating depressive-like behaviors.

CRMP2 is a therapeutic target that suppresses the aggressiveness of breast cancer cells by stabilizing RECK

Metastatic breast cancer is characterized by high mortality and limited therapeutic target. During tumor metastasis, cytoskeletal reorganization is one of the key steps in the migration and invasion of breast cancer cells. Collapsin response mediator protein 2 (CRMP2) is a cytosolic phosphoprotein that plays an important role in regulating cytoskeletal dynamics. Previous researches have reported that altered CRMP2 expression is associated with breast cancer progression, but the underlying mechanism remains poorly understood. Here, we show that CRMP2 expression is reduced in various subtypes of breast cancers and negatively correlated with lymphatic metastasis. Overexpression of CRMP2 significantly inhibits invasion and stemness in breast cancer cells, while downregulation of CRMP2 promotes cell invasion, which is not required for tubulin polymerization. Mechanistic studies demonstrate that CRMP2 interacts with RECK, prevents RECK degradation, which, in turn, blocks NF-κB and Wnt signaling pathways. Furthermore, we find that phosphorylation of CRMP2 at T514 and S522 remarkably abolishes its functions to bind with RECK and to inhibit cell invasion. Pharmacologic rescue of CRMP2 expression suppressed breast cancer metastasis in vitro and in vivo and stimulated a synergetic effect with FN-1501 that induces CRMP2 dephosphorylation. Collectively, this study highlights the potential of CRMP2 as a therapeutic target in breast cancer metastasis and reveals a distinct mechanism of CRMP2.

Regulation of Adipogenesis and Lipid Deposits by Collapsin Response Mediator Protein 2.

As emerging evidence suggesting neurodegenerative diseases and metabolic diseases have common pathogenesis, we hypothesized that the neurite outgrowth-controlling collapsin response mediator protein 2 (CRMP2) was involved in energy homeostasis. Therefore, putative roles of CRMP2 in adipocyte differentiation (adipogenesis) and lipid metabolism were explored and addressed in this study. CRMP2 expression profiles were in vitro and in vivo characterized during adipogenic process of 3T3-L1 pre-adipocytes and diet-induced obese (DIO) mice, respectively. Effects of CRMP2 on lipid metabolism and deposits were also analyzed. Our data revealed that CRMP2 expression pattern was coupled with adipogenic stages. CRMP2 overexpression inhibited cell proliferation at MCE phase, and significantly reduced lipid contents by down-regulating adipogenesis-driving transcription factors and lipid-synthesizing enzymes. Interestingly, GLUT4 translocation and the lipid droplets fusion were disturbed in CRMP2-silencing cells by affecting actin polymerization. Moreover, adipose CRMP2 was significantly increased in DIO mice, indicating CRMP2 is associated with obesity. Accordingly, CRMP2 exerts multiple functions in adipogenesis and lipid deposits through mediating cell proliferation, glucose/lipid metabolism and cytoskeleton dynamics. The present study identifies novel roles of CRMP2 in mediating adipogenesis and possible implication in metabolic disorders, as well as provides molecular evidence supporting the link of pathogenesis between neurodegenerative diseases and metabolic abnormalities.

CRMP2 improves memory deficits by enhancing the maturation of neuronal dendritic spines after traumatic brain injury

Our recent study investigated the role of collapsin response mediator protein-2 (CRMP2) on dendritic spine morphology and memory function after traumatic brain injury (TBI). First, we examined the density and morphology of dendritic spines in Thy1-GFP mice on the 1 st day (P1D) and 7th day (P7D) after controlled cortical impact injury (CCI). The dendritic spine density in the hippocampus was decreased on P1D, in which mainly mushroom-type and thin-type spines were lost. The density of dendritic spines was increased on P7D, most of which were of the thin type. Next, we explored the expression of CRMP2 on P1D and P7D. CRMP2 expression was decreased on P1D, but the levels of the CRMP2 breakdown product were increased. On P7D, the expression pattern was the opposite. Then, we constructed CRMP2 overexpression and knockdown plasmids and transfected them into cultured neurons in vitro. CRMP2 increased the dendritic spine density of cultured neurons and the proportion of mushroom-type spines, while CRMP2-shRNA reduced the dendritic spine density and the proportion of mushroom-type spines. To determine the role of CRMP2 in dendritic spines after TBI, we stereotactically injected the CRMP2 overexpression and knockdown viruses into the hippocampus and found that CRMP2 increased the dendritic spine density and the proportion of mushroom-type spines after TBI. Meanwhile, as suggested by the morphological changes, fear conditioning behavioral experiments confirmed that CRMP2 improved memory deficits after TBI.

Dynamic CRMP2 Regulation of CaV2.2 in the Prefrontal Cortex Contributes to the Reinstatement of Cocaine Seeking.

Cocaine addiction remains 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.

CRMP2-derived peptide ST2-104 (R9-CBD3) protects SH-SY5Y neuroblastoma cells against Aβ25-35-induced neurotoxicity by inhibiting the pCRMP2/NMDAR2B signaling pathway

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.

Expression of collapsin response mediator protein 2 in visual cortex of form deprivation amblyopic rats

Objective To study the expression of collapsin response mediator protein 2 (CRMP-2) in the visual cortex of monocular form deprivation amblyopia rats. Methods Sixty-four 14-day-old rats were randomly divided into monocular deprivation amblyopia group and normal control group by random number table method.Right eyelid margin suture was performed at 14 days after birth in the monocular deprivation amblyopia group.Eight rats in the monocular deprivation amblyopia group and the normal control group were observed at 14, 21, 45 and 120 days after birth, respectively.Flash visual evoked potential (F-VEP) was used to dectect the latency and amplitude of P1 wave.The expression of CRMP-2 in visual cortex was observed by immunohistochemical method.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of the University of South China (No.20140228). Results F-VEP results showed that the amplitudes of P1 were decreased and latent periods of P1 were prolonged in the monocular deprivation amblyopia group compared with the normal control group (t=16.760, P=0.000; t=-22.919, P=0.000). CRMP-2 expression levels in the visual cortex of monocular deprivation amblyopia groups and normal control groups were compared at different time points after birth, and the differences were statistically significant (Fgroup=8.855, P=0.010; Ftime=63.091, P=0.000). Compared with normal control groups, the expressions of CRMP-2 at the postnatal 21, 45 and 120 days were obviously decreased in the monocular deprivation amblyopia groups, the differences were statistically significant (all at P<0.05). Conclusions CRMP-2 may be involved in the occurrence and development of amblyopia. Key words: Collapsin response mediator protein 2; Form deprivation amblyopia; Rat; Visual cortex

Neuronal Conditional Knockout of Collapsin Response Mediator Protein 2 Ameliorates Disease Severity in a Mouse Model of Multiple Sclerosis.

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.

Induction of CRMP-2 phosphorylation by CDK5 restricts the repair of damaged optic nerve.

To study the mechanism of collapsin response mediator protein-2 (CRMP-2) phosphorylation changes and cyclin-dependent kinase 5 (CDK5) expression after optic nerve injury. Optic nerve injury rat models were constructed, the messenger RNA (mRNA) level of CRMP-2 in optic nerve tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after building models 0, 3, 7, and 14 days. The protein expression of CRMP-2, phospho-CRMP-2 (p-CRMP-2), and CDK5 were also determined by western blot analysis. Lentivirus overexpressing CRMP-2 and CRMP-2 small interfering RNA (siRNA) plasmid were designed and transfected to retina ganglion cells (RGCs), and then the neurites outgrowth of RGCs were cultured with CDK5 inhibitor or CDK5 activator was determined by tubulin staining. Inhibition on CDK5 promotes injured optic nerve by using carrying CDK5 siRNA inject into vitreous chamber. There was no significant change in CRMP-2 expression in optic nerve injury rat, while p-CRMP-2 expression was evidently increased compared with sham operation group. The expression level of CDK5 in optic nerve tissue was upregulated after optic nerve injury in rat, and the upward trend of p-CRMP-2 and CDK5 was consistent with the time after the injury was prolonged. Inhibition on CDK5 evidently decreased the expression of p-CRMP-2. CDK5 siRNA had an obvious repair effect on the injured optic nerve. The increase of CDK5 activity can lead to CRMP-2 hyperphosphorylation, which results in the difficult repair of damaged optic nerve. Therefore, inhibition on CDK5 could promote the repair of damaged optic nerve.

Cdk5-mediated CRMP2 phosphorylation is necessary and sufficient for peripheral neuropathic pain

Neuropathic pain results from nerve injuries that cause ectopic firing and increased nociceptive signal transmission due to activation of key membrane receptors and channels. The dysregulation of trafficking of voltage-gated ion channels is an emerging mechanism in the etiology of neuropathic pain. We identify increased phosphorylation of collapsin response mediator protein 2 (CRMP2), a protein reported to regulate presynaptic voltage-gated calcium and sodium channels. A spared nerve injury (SNI) increased expression of a cyclin dependent kinase 5 (Cdk5)-phosphorylated form of CRMP2 in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) in the ipsilateral (injured) versus the contralateral (non-injured) sites. Biochemical fractionation of spinal cord from SNI rats revealed the increase in Cdk5-mediated CRMP2 phosphorylation to be enriched to pre-synaptic sites. CRMP2 has emerged as a central node in assembling nociceptive signaling complexes. Knockdown of CRMP2 using a small interfering RNA (siRNA) reversed SNI-induced mechanical allodynia implicating CRMP2 expression as necessary for neuropathic pain. Intrathecal expression of a CRMP2 resistant to phosphorylation by Cdk5 normalized SNI-induced mechanical allodynia, whereas mimicking constitutive phosphorylation of CRMP2 resulted in induction of mechanical allodynia in naïve rats. Collectively, these results demonstrate that Cdk5-mediated CRMP2 phosphorylation is both necessary and sufficient for peripheral neuropathic pain.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

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.

CRMP2 Phosphorylation Drives Glioblastoma Cell Proliferation

Glioblastoma (GBM) is an aggressive primary brain tumor. The rapid growth and the privileged provenance of the tumor within the brain contribute to its aggressivity and poor therapeutic targeting. A poor prognostic factor in glioblastoma is the deletion or mutation of the Nf1 gene. This gene codes for the protein neurofibromin, a tumor suppressor gene that is known to interact with the collapsin response mediator protein 2 (CRMP2). CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 have recently been implicated in cancer progression. The CRMP2-neurofibromin interaction protects CRMP2 from its phosphorylation by cyclin-dependent kinase 5 (Cdk5), an event linked to cancer progression. In three human glioblastoma cell lines (GL15, A172, and U87), we observed an inverse correlation between neurofibromin expression and CRMP2 phosphorylation levels. Glioblastoma cell proliferation was dependent on CRMP2 expression and phosphorylation by Cdk5 and glycogen synthase kinase 3 beta (GSK3β). The CRMP2 phosphorylation inhibitor (S)-lacosamide reduces, in a concentration-dependent manner, glioblastoma cell proliferation and induced apoptosis in all three GBM cell lines tested. Since (S)-lacosamide is bioavailable in the brain, we tested its utility in an in vivo orthotopic model of GBM using GL261-LucNeo glioma cells. (S)-lacosamide decreased tumor size, as measured via in vivo bioluminescence imaging, by ~54% compared to vehicle control. Our results introduce CRMP2 expression and phosphorylation as a novel player in GBM proliferation and survival, which is enhanced by loss of Nf1.

Minocycline restores cognitive-relative altered proteins in young bile duct-ligated rat prefrontal cortex

AimsBile duct ligation (BDL) model is used to study hepatic encephalopathy accompanied by cognitive impairment. We employed the proteomic analysis approach to evaluate cognition-related proteins in the prefrontal cortex of young BDL rats and analyzed the effect of minocycline on these proteins and spatial memory. Main methodsBDL was induced in young rats at postnatal day 17. Minocycline as a slow-release pellet was implanted into the peritoneum. Morris water maze test and two-dimensional liquid chromatography-tandem mass spectrometry were used to evaluate spatial memory and prefrontal cortex protein expression, respectively. We used 2D/LC-MS/MS to analyze for affected proteins in the prefrontal cortex of young BDL rats. Results were verified with Western blotting, immunohistochemistry, and quantitative real-time PCR. The effect of minocycline in BDL rats was assessed. Key findingsBDL induced spatial deficits, while minocycline rescued it. Collapsin response mediator protein 2 (CRMP2) and manganese-dependent superoxide dismutase (MnSOD) were upregulated and nucleoside diphosphate kinase B (NME2) was downregulated in young BDL rats. BDL rats exhibited decreased levels of brain-derived neurotrophic factor (BDNF) mRNA as compared with those by the control. However, minocycline treatment restored CRMP2 and NME2 protein expression, BDNF mRNA level, and MnSOD activity to control levels. SignificanceWe demonstrated that BDL altered the expression of CRMP2, NME2, MnSOD, and BDNF in the prefrontal cortex of young BDL rats. However, minocycline treatment restored the expression of the affected mediators that are implicated in cognition.

Silencing collapsin response mediator protein-2 reprograms macrophage phenotype and improves infarct healing in experimental myocardial infarction model.

BackgroundDelayed M1 toward M2 macrophage phenotype transition is considered one of the major causes for the impaired healing after myocardial infarction (MI). While searching for molecules that modulate M1 and M2 macrophage polarization, we identified collapsin response mediator protein-2 (CRMP2) as a novel molecule involved in macrophage polarization to M1. In this study, we evaluated the effect of silencing CRMP2 on macrophage polarization, inflammation and fibrosis post myocardial infarction.MethodsCRMP2 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry, quantitative real-time PCR (qPCR), Western blotting or immunohistochemistry. CRMP2 siRNA was delivered into the macrophages infiltrated in the wound of ApoE−/− mice through lipidoid nanoparticle, and fibrosis, leukocyte infiltration and inflammation parameters were measured with qPCR. Infarct size was measured with Masson’s trichrome staining. Echocardiography was performed to assess ventricular systolic dimension, left ventricular diastolic dimension, anterior wall thickness and posterior wall thickness. Student’s t-test (for 2 groups) and ANOVA (for > 2 groups) were used for statistical analyses.ResultsCRMP2 was expressed in a higher level in M1 macrophages than M2 subsets, and CRMP2 RNA interference (RNAi) resulted in a switch of bone marrow-derived macrophages from M1 to M2 phenotype. High level of CRMP2 was also observed in the macrophages infiltrated in the infarct area 3 days post MI in both wildtype (WT) and ApoE−/− mice, and the expression of CRMP2 retained in the infiltrated macrophages of ApoE−/− mice but not in that of WT mice 10 days after MI. Nanoparticle-mediated delivery of CRMP2 siRNA to ApoE−/− mice with MI resulted in dramatic switch of wound macrophages from M1 to M2 phenotype, marked decrease in inflammation and fibrosis, and significant attenuation of post-MI heart failure and mortality.ConclusionCRMP2 is highly expressed in M1 macrophages and silencing CRMP2 reprograms macrophage phenotype and improves infarct healing in atherosclerotic mice.

Identification of biomarkers for endometriosis in eutopic endometrial cells from patients with endometriosis using a proteomics approach

Endometriosis is a gynecological disease defined as the presence of endometrial tissue outside the uterine cavity, which is caused by various factors. Proteomic analysis of two sets of eutopic endometrial cells collected from the menstrual blood of females with (n=6; n=3) or without (n=6; n=3) endometriosis was performed to identify novel potential biomarkers for endometriosis. The data revealed that samples from endometriosis patients had stem cell characteristics, as they had higher mRNA expression levels of octamer-binding transcription factor 4 (Oct-4), C-X-C chemokine receptor type 4 (CXCR4), SRY-box containing gene2 (SOX2) and mesenchymal-epithelial transition factor (MET) compared with that of the normal controls. Three proteins, collapsin response mediator protein 2 (CRMP2), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) and myosin regulatory light polypeptide 9 (MYL9), were simultaneously identified from the two sets of samples from females with or without endometriosis by two-dimensional electrophoresis (2-DE). A difference in CRMP2 expression was confirmed with western blotting. Taken together, the results suggest that CRMP2 plays a role in the pathogenesis of endometriosis.

Human T Lymphotropic Virus Type 1 Increases T Lymphocyte Migration by Recruiting the Cytoskeleton Organizer CRMP2

Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuroinflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1-induced migration seems, in part, supported by the ability of infected cell to increase the proteosomal degradation of short CRMP2. Finally, gene expression in CD69(+) cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1-infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation.

Voltage-gated calcium channels are not affected by the novel anti-epileptic drug lacosamide

The novel anti-epileptic drug lacosamide targets two proteins - voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP-2) - suggesting dual modes of action for lacosamide. We recently identified the neurite outgrowth and axonal guidance protein CRMP-2 as a novel partner and regulator of the presynaptic N-type voltage-gated Ca(2+) channel (CaV2.2) [Brittain et al., J. Biol. Chem. 284: 31375-31390 (2009)]. Here we examined the effects of lacosamide on voltage-gated Ba(2+) channels. Lacosamide did not affect Ba(2+) currents via N- and P/Q- channels in rat hippocampal neurons or L-type Ca(2+) channels in a mouse CNS neuronal cell line, respectively. N-type Ba(2+) currents, augmented by CRMP-2 expression, were also unaffected by acute or chronic lacosamide exposure. These results establish that the anti-epileptic mode of action of lacosamide does not involve these voltage-gated Ca(2+) channels.