Collapsin Response Mediator Proteins of Neonatal Rat Brain Interact with Chondroitin Sulfate

Abstract

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 immunohistochemically 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. The extracellular matrix (ECM)1 of the developing brain has a unique composition from lecticans, tenascins, laminins, and hyaluronic acid (1-5). Lecticans are proteoglycans that carry chondroitin sulfate (CS) side chains on core proteins encompassing a N-terminal hyaluronan binding domain and a Cterminal lectin domain. Four different members of the lectican protein family are known (neurocan, aggrecan, versican, and brevican) (1). Their multidomain composition enables lecticans to interact with multiple cell surface molecules and diffusible ligands (see below) (6, 7). Chondroitin sulfates are glycosaminoglycans composed of repeated glucuronic acid-[␤1,3]-Nacetylgalactosamine disaccharide units that are linked by [␤1,4]-glycosidic bonds into long unbranched chains with molecular masses of up to 50 kDa and more (8). With respect to the position of sulfate esters at the galactosamine, CSA (C-4-S, containing C-4-sulfate) and CSC (C-6-S, containing C-6-sulfate) are distinguished. The expression of these proteoglycan core proteins is precisely tuned during brain development (9). Moreover, the disaccharide composition of CS is regulated (9, 10).The functions of chondroitin sulfate proteoglycans (CS-PGs) in neural tissue can be categorized into effects on cell adhesion, cell migration, neurite formation, neuron polarization, synaptic modulation/plasticity, and neuron survival (for reviews, see Ref. 6 and references therein and Ref. 11). These effects often critically depend on glycosa...

Cell Death as a Regulator of Cerebellar Histogenesis and Compartmentation

“…If we may speculate here, it is conceivable that PC death leads to the liberation of intracellular molecules that act as repulsive signals in the extracellular matrix. Interestingly, collapsin response mediator protein liberation has been postulated to occur subsequent to cell death [138]. Direct experimental testing of this hypothesis will ultimately require abrogation of midline PC death, and analysis of climbing fiber dynamics during the critical developmental phase defined here.…”

Calpain-Cleaved Collapsin Response Mediator Protein-3 Induces Neuronal Death after Glutamate Toxicity and Cerebral Ischemia

“…Antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Advanced Immunochemicals (Long Beach, CA). Antibodies to CRMP-1, CRMP-2, CRMP-3, and CRMP-4 have been described previously (Franken et al, 2003) and were used at 1:10,000 for Western blotting.…”

Semaphorin 3A Stimulates Neurite Extension and Regulates Gene Expression in PC12 Cells

“…The samples were seperated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schü ll), and Western blots were developed using the Vectastain ABC kit (Vector Laboratories) according to manufactor's instructions. The following primary antibodies were used: anti-Sema5A (1:2000), anti-Crmp2 (1:1000), and antiCrmp4 (1:1000) (17). The anti-Sema5A antibody was raised against the C-terminal 130 amino acids expressed in bacteria using the expression vector pQE-30 (Qiagen).…”