Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.     

Subcellular localization of collapsin response mediator proteins to lipid rafts

Collapsin response mediator proteins (CRMPs) are involved in signal transduction after exposure of neural cells to the axon guidance molecule Semaphorin 3A/collapsin. All five known CRMPs are expressed in the developing cerebral cortex and neocortical neurons are responsive to Semaphorin 3A. Here, we examine the expression and subcellular localization of CRMPs in neocortical neurons and in neonatal rat brain. In neocortical neurons CRMP-4 was detected in the perikaryon with a diffuse cytosolic distribution. In neurites and at growth cones punctate staining patterns were observed. Extraction of neuron cultures with methyl-beta-cyclodextrin to deplete cholesterol caused rapid redistribution of the punctate CRMP-4 staining into larger patches and abundant growth cone collapse. Western blotting of brain extracts demonstrated for all CRMPs the existence of soluble, detergent-extractable, and Triton X-100-resistant forms. Furthermore, sucrose density gradient centrifugation after solubilization of brain membranes with Triton X-100 revealed that CRMP-1, -3, -5, and to a lower extent CRMP-4 are associated with a detergent-resistant fraction with low buoyant density, but CRMP-2 was not detectable in this fraction. Thus, we propose that lipid rafts form sites for the compartmentalization of signaling events involving specific CRMPs and that the integrity of these membrane microdomains is essential for the maintenance of growth cones.     

Proteomic analysis of neonatal mouse brain: evidence for hypoxia- and ischemia-induced dephosphorylation of collapsin response mediator proteins

Perinatal hypoxia and ischemia (HI) are a significant cause of mortality and morbidity. To understand the molecular mechanisms for HI-induced brain damage, here we used a proteomic approach to analyze the alteration and modification of proteins in neonatal mouse brain 24 h after HI treatment. Significant changes of collapsin response mediator proteins (CRMPs) were observed in HI brain. CRMPs are a family of cytosolic proteins involved in axonal guidance and neuronal outgrowth. We found that CRMP2, CRMP4 and CRMP5 proteins were altered post-translationally after HI treatment. Mass spectrometric and Western blot analyses detected hypophosphorylated CRMP proteins after HI. Further analysis of CRMP kinases indicated inactivation of cyclin dependent kinase 5 (CDK5), a priming kinase of CRMPs and a neuronal specific kinase that plays pivotal roles in neuronal development and survival. The reduction of CDK5 activity was associated with underexpression of its activator p35. Taken together, our findings reveal HI-induced dephosphorylation of CRMPs in neonatal brain and suggest a novel mechanism for this modification. Hypophosphorylated CRMPs might be implicated in the pathogenesis of HI-related neurological disorders.     

Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain

Neurofibromin, encoded by the neurofibromatosis type 1 (NF1) gene, regulates the Ras and cAMP pathways and plays a role in proliferation and neuronal morphogenesis. The details of the molecular mechanism of neurofibromin action in these processes are still unclear. In this study, immunoprecipitation and proteomics were used to identify novel proteins from rat brain that interact with neurofibromin. Mass spectrometry analysis showed that two proteins, the collapsin response mediator protein-2 (CRMP-2) and propionyl-CoA carboxylase alpha chain (PCCA), associated with neurofibromin. Immunoprecipitation-immunoblotting analysis confirmed the interactions between neurofibromin and CRMP-2 and CRMP-4, but not CRMP-1, in rat brain. CDK5, a kinase that regulates CRMP-2 in axonal outgrowth, was required for the interaction between neurofibromin and CRMP-2. Since both neurofibromin and CRMP proteins are involved in proliferation and axonal morphogenesis, these results suggest that the interaction with CRMPs contributes to the function of neurofibromin in tumorigenesis and neuronal morphogenesis.     

Fyn promotes phosphorylation of collapsin response mediator protein 1 at tyrosine 504, a novel,isoform‐specific regulatory site

In vertebrates the collapsin response mediator proteins (CRMPs) are encoded by five highly related genes. CRMPs are cytosolic phosphoproteins abundantly expressed in developing and mature mammalian brains. CRMPs are best understood as effectors of Semaphorin 3A signaling regulating growth cone collapse in migratory neurons. Phosphorylation in the carboxyl‐terminal regulatory domain of CRMPs by several serine/threonine kinases has been described. These phoshorylation events appear to function, at least in part, to disrupt the interaction of CRMPs with tubulin heterodimers. In a large‐scale phosphoproteomic analysis of murine brain, we recently identified a number of in vivo tyrosine phosphorylation sites on CRMP isoforms. Using biochemical approaches and quantitative mass spectrometry we demonstrate that one of these sites, CRMP1 tyrosine 504 (Y504), is a primary target of the Src family of tyrosine kinases (SFKs), specifically Fyn. Y504 is adjacent to CDK5 and GSK‐3β sites that regulate the interaction of CRMPs with tubulin. Although Y504 is highly conserved among vertebrate CRMP1 orthologs, a residue corresponding to Y504 is absent in CRMP isoforms 2–5. This suggests an isoform‐specific regulatory role for CRMP1 Y504 phosphorylation and may help explain the observation that CRMP1‐deficient mice exhibit neuronal migration defects not compensated for by CRMPs 2–5. J. Cell. Biochem. 111: 20–28, 2010. © 2010 Wiley‐Liss, Inc.     

The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo

The position of the centrosome ahead of the nucleus has been considered crucial for coordinating neuronal migration in most developmental situations. The proximity of the centrosome has also been correlated with the site of axonogenesis in certain differentiating neurons. Despite these positive correlations, accumulating experimental findings appear to negate a universal role of the centrosome in determining where an axon forms, or in leading the migration of neurons. To further examine this controversy in an in vivo setting, we have generated cell type-specific multi-cistronic gene expression to monitor subcellular dynamics in the developing zebrafish cerebellum. We show that migration of rhombic lip-derived neurons is characterized by a centrosome that does not persistently lead the nucleus, but which is instead regularly overtaken by the nucleus. In addition, axonogenesis is initiated during the onset of neuronal migration and occurs independently of centrosome proximity. These in vivo data reveal a new temporal orchestration of organelle dynamics and provide important insights into the variation in intracellular processes during vertebrate brain differentiation.     

Collapsin response mediator protein-2 accelerates axon regeneration of nerve-injured motor neurons of rat

The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1-5). The functional consequence of CRMP-2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP-2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP-1, CRMP-2, CRMP-5 mRNA expressions increased after nerve injury, whereas CRMP-3 and CRMP-4 mRNA did not show any significant change. In the N1E-115 cells, CRMP-2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP-2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP-2 (DeltaC381), which acts as a dominant negative form of CRMP-2, inhibited neurite formation. Collectively, it would be plausible that CRMP-2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP-2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde-tracer, Fluoro-Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP-2-overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP-2 has potent neurite elongation activity in nerve regeneration in vivo.     

Collapsin response mediator proteins (CRMPs)

The members of the collapsin response mediator protein (CRMP) family—five cytosolic phosphoproteins—are highly expressed throughout brain development. The first member to be cloned, CRMP2, was identified as an intracellular messenger required for the growth cone-collapse induced by semaphorin 3A (Sema3A). A rapidly expanding body of study indicates that the functions of CRMPs are not solely limited to the signaling transduction of the Sema3A guidance cue. They are probably involved in multiple cellular and molecular events involved in apoptosis/proliferation, cell migration, and differentiation. In the adult brain, the expression of CRMPs is dramatically downregulated. However, they remain expressed in structures that retain their capacity for differentiation and plasticity and also in a subpopulation of oligodendrocytes (CRMP2 and CRMP5). Moreover, the expression of CRMPs is altered in neurodegenerative diseases, and these proteins may be of key importance in the physiopathology of the adult nervous system.     

Collapsin response mediator proteins (CRMPs): involvement in nervous system development and adult neurodegenerative disorders

The members of the collapsin response mediator protein (CRMP) family-five cytosolic phosphoproteins -are highly expressed throughout brain development. The first member to be cloned, CRMP2, was identified as an intracellular messenger required for the growth cone-collapse induced by semaphorin 3A (Sema3A). A rapidly expanding body of study indicates that the functions of CRMPs are not solely limited to the signaling transduction of the Sema3A guidance cue. They are probably involved in multiple cellular and molecular events involved in apoptosis/proliferation, cell migration, and differentiation. In the adult brain, the expression of CRMPs is dramatically downregulated. However, they remain expressed in structures that retain their capacity for differentiation and plasticity and also in a subpopulation of oligodendrocytes (CRMP2 and CRMP5). Moreover, the expression of CRMPs is altered in neurodegenerative diseases, and these proteins may be of key importance in the physiopathology of the adult nervous system.     

Collapsin Response Mediator Proteins Regulate Neuronal Development and Plasticity by Switching Their Phosphorylation Status

Collapsin response mediator protein (CRMP) was originally identified as a molecule involved in semaphorin3A signaling. CRMPs are now known to consist of five homologous cytosolic proteins, CRMP1–5. All of them are phosphorylated and highly expressed in the developing and adult nervous system. In vitro experiments have clearly demonstrated that CRMPs play important roles in neuronal development and maturation through the regulation of their phosphorylation. Several recent knockout mice studies have revealed in vivo roles of CRMPs in neuronal migration, neuronal network formation, synapse formation, synaptic plasticity, and neuronal diseases. Dynamic spatiotemporal regulation of phosphorylation status of CRMPs is involved in many aspects of neuronal development.     

CRMP5 regulates generation and survival of newborn neurons in olfactory and hippocampal neurogenic areas of the adult mouse brain

The Collapsin Response Mediator Proteins (CRMPS) are highly expressed in the developing brain, and in adult brain areas that retain neurogenesis, ie: the olfactory bulb (OB) and the dentate gyrus (DG). During brain development, CRMPs are essentially involved in signaling of axon guidance and neurite outgrowth, but their functions in the adult brain remain largely unknown. CRMP5 has been initially identified as the target of auto-antibodies involved in paraneoplasic neurological diseases and further implicated in a neurite outgrowth inhibition mediated by tubulin binding. Interestingly, CRMP5 is also highly expressed in adult brain neurogenic areas where its functions have not yet been elucidated. Here we observed in both neurogenic areas of the adult mouse brain that CRMP5 was present in proliferating and post-mitotic neuroblasts, while they migrate and differentiate into mature neurons. In CRMP5(-/-) mice, the lack of CRMP5 resulted in a significant increase of proliferation and neurogenesis, but also in an excess of apoptotic death of granule cells in the OB and DG. These findings provide the first evidence that CRMP5 is involved in the generation and survival of newly generated neurons in areas of the adult brain with a high level of activity-dependent neuronal plasticity.     

Brain CRMP Forms Heterotetramers Similar to Liver Dihydropyrimidinase

Abstract: The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin-1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two-hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two-hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two-hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8–134 and 281–435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.     

Calpain‐truncated CRMP‐3 and ‐4 contribute to potassium deprivation‐induced apoptosis of cerebellar granule neurons

Increasing evidence shows that calpain‐mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain‐mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation‐induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2‐D DIGE and MALDI‐TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP‐1, ‐2, ‐3 and ‐4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain‐mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP‐1, ‐2 and ‐4 were truncated by calpain at the C‐terminus, whereas CRMP‐3 was cleaved at the N‐terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation‐triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP‐3 and ‐4, but not CRMP‐1 and ‐2, significantly induced neuronal apoptosis. These findings demonstrated that calpain‐truncated CRMP‐3 and ‐4 act as pro‐apoptotic players when CGNs undergo apoptosis.     

Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord

Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co‐injection of phosphorylation‐mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 911–920, 2013     

Identification of CRAM, a novel unc-33 gene family protein that associates with CRMP3 and protein-tyrosine kinase(s) in the developing rat brain

Four members of collapsin response mediator proteins (CRMPs) are thought to be involved in the semaphorin-induced growth cone collapse during neural development. Here we report the identification of a novel CRMP3-associated protein, designated CRAM for CRMP3-associated molecule, that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids, shows 57% identity with dihydropyrimidinase, and shows 50-51% identity with CRMPs. CRAM appears to form a large complex composed of CRMP3 and other unidentified proteins in vivo. Indeed, CRAM physically associates with CRMP3 when co-expressed in COS-7 cells. The expression of CRAM is brain-specific, is high in fetal and neonatal rat brain, and decreases to very low levels in adult brain. Moreover, CRAM expression is up-regulated during neuronal differentiation of embryonal carcinoma P19 and PC12 cells. Finally, immunoprecipitation analysis of rat brain extracts shows that CRAM is co-immunoprecipitated with proteins that contain protein-tyrosine kinase activity. Taken together, our results suggest that CRAM, which interacts with CRMP3 and protein-tyrosine kinase(s), is a new member of an emerging family of molecules that potentially mediate signals involved in the guidance and outgrowth of axons.     

Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.