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The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.

The chick axon-associated surface glycoprotein neurofascin is implicated in axonal growth and fasciculation as revealed by antibody perturbation experiments. Here we report the complete cDNA sequence of neurofascin. It is composed of four structural elements: At the NH2 terminus neurofascin contains six Ig-like motifs of the C2 subcategory followed by four fibronectin type III (FNIII)-related repeats. Between the FNIII-like repeats and the plasma membrane spanning region neurofascin contains a domain 75-amino acid residues-long rich in proline, alanine and threonine which might be the target of extensive O-linked glycosylation. A transmembrane segment is followed by a 113-amino acid residues-long cytoplasmic domain. Sequence comparisons indicate that neurofascin is most closely related to chick Nr-CAM and forms with L1 (Ng-CAM) and Nr-CAM a subgroup within the vertebrate Ig superfamily. Sequencing of several overlapping cDNA probes reveals interesting heterogeneities throughout the neurofascin polypeptide. Genomic Southern blots analyzed with neurofascin cDNA clones suggest that neurofascin is encoded by a single gene and its pre-mRNA might be therefore alternatively spliced. Northern blot analysis with domain specific probes showed that neurofascin mRNAs of about 8.5 kb are expressed throughout development in embryonic brain but not in liver. Isolation of neurofascin by immunoaffinity chromatography results in several molecular mass components. To analyze their origin the amino-terminal sequences of several neurofascin components were determined. The NH2-terminal sequences of the 185, 160, and 110-135 kD components are all the same as the NH2 termini predicted by the cDNA sequence, whereas the other neurofascin components start with a sequence found in a putative alternatively spliced segment between the Ig- and FNIII-like part indicating that they are derived by proteolytic cleavage. A combination of enzymatic and chemical deglycosylation procedures and the analysis of peanut lectin binding reveals O- and N-linked carbohydrates on neurofascin components which might generate additional heterogeneity.

CALEB/NGC is a neural member of the epidermal growth factor protein family expressed in axon and synapse-rich areas of the nervous system and shown to be important for neurite formation. It can bind to the extracellular matrix proteins tenascin-R and tenascin-C. Here we show that CALEB/NGC interacts with the Golgi-associated protein PIST. PIST was originally described as an interaction partner of the small GTPase TC10 and was then found to be Golgi-associated by binding to syntaxin-6 and to be important for the transport of frizzled proteins and the cystic fibrosis transmembrane conductance regulator to the plasma membrane. In addition, PIST was demonstrated to be involved in autophagy and linked to processes of neurodegeneration. CALEB/NGC interacts with PIST in the yeast two-hybrid system. This interaction can be confirmed by co-immunoprecipitations and co-localization studies. The juxtamembrane cytoplasmic peptide segment of CALEB/NGC, highly conserved during evolution, mediates the binding to PIST. CALEB/NGC co-localizes with PIST in the Golgi apparatus of transfected COS7 cells and in Golgi-derived vesicles after brefeldin A or nocodazole treatment. Co-localization studies in primary hippocampal cells and analysis of Purkinje cells of colchicine-treated rats, serving as an in vivo model system to block microtubule-dependent transport processes, support the view that PIST is an interaction partner of CALEB/NGC and implicate that this interaction may play a role in the intracellular transport of CALEB/NGC.