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.     

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.     

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.     

Expression of collapsin response mediator proteins in the nervous system of embryonic zebrafish

Collapsin response mediator proteins (CRMPs also known as TUC, Drp, Ulip, TOAD-64) are cytosolic phosphoproteins that are involved in signal transduction during axon growth and in cytoskeletal dynamics. Here we report cloning and mRNA expression patterns of CRMP-1, -2, -3, -4 and, owing to a genome duplication in teleosts, two homologs of CRMP-5 (CRMP-5a and -5b) in embryonic zebrafish at 16 and 24 h post-fertilization (hpf). CRMPs are evolutionarily conserved and zebrafish CRMPs show amino acid identities of 76–90% with their homologs in humans, with the exception of CRMP-3, which shows only 67% homology. Between 16 and 24 hpf, expression of CRMPs generally increased in many regions of the CNS undergoing neuronal differentiation and axonogenesis, but not in the proliferative ventricular zone. Structures that were typically labeled by most, but not all the CRMP probes were the telencephalon, the nucleus of the tract of the post-optic commissure, the epiphysis, the nucleus of the medial longitudinal fascicle, clusters of hindbrain neurons, cranial ganglia, as well as Rohon-Beard neurons. No expression of CRMP mRNAs was observed outside the nervous system. Thus, expression patterns of different CRMP family members correlate with neuronal differentiation and axonogenesis in embryonic zebrafish.     

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.     

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 protein-3/unc-33-like protein-4 gene: organization, chromosomal mapping and expression in the developing mouse brain

CRMPs (collapsin response mediator proteins)/ULIPs (unc-33-like proteins) are a family of intracytoplasmic proteins that are expressed mainly in the brain. The involvement of CRMP/ULIP members in neuronal differentiation, growth cone motility and axonal collapse has been suggested. We recently found that a member of this family, CRMP3/ULIP4, corresponds to POP66 (paraneoplastic oligodendrocyte protein of 66 kDa), a protein which may be associated with auto-immune induced-neuronal degeneration in paraneoplastic neurological syndromes. However, the physiological functions of these proteins remain to be elucidated. Further studies, including the generation of cell lines and of animals with modified/disrupted CRMP/ULIP gene expression, are necessary to explore the functions of this protein. We have cloned and determined the organization and chromosomal localization of the mouse gene encoding CRMP3/ULIP4. The gene is composed of 14 exons and spans more than 20 kb. We assigned the mouse CRMP3/ULIP4 gene to the distal end of chromosome 7. In mouse brain, in situ hybridization showed that CRMP3/ULIP4 mRNA is expressed mainly in the dentate gyrus of hippocampus, in the granular layers of cerebellum and in the inferior olive of the pons, the nucleus which controls movement and posture, and adjusts the major output of descending motor system.     

Critical role of collapsin response mediator protein-associated molecule CRAM for filopodia and growth cone development in neurons

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.     

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.     

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.     

Molecular characterization of CRMP5, a novel member of the collapsin response mediator protein family

The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49-50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68-75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks.     

Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5(-/-) cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.     

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.     

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.     

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.     

Collapsin response mediator proteins (CRMPs) are a new class of microtubule-associated protein (MAP) that selectively interacts with assembled microtubules via a taxol-sensitive binding interaction

Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3β (GSK3β) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3β. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

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.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

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.