Homophilic interactions of chick neurofascin in trans are important for neurite induction

Abstract

Neurofascin is a member of the immunoglobulin superfamily involved in axon extension and fasciculation. Here we apply adenoviral short hairpin RNA (shRNA) expression in primary neurons, PC12-NIH/3T3 co-cultures in combination with Luminex assays, to demonstrate homophilic interactions of neurofascin for neurite outgrowth. An adenoviral vector was constructed for the expression of shRNA in primary tectal cells that inhibits gene expression similar to short interfering RNA. We demonstrate that after shRNA-mediated knockdown neuronal neurofascin expression is important for neurite outgrowth on a neurofascin substrate. Neurite outgrowth assays reveal that neurite formation of PC12 cells is increased when neurofascin is overexpressed on both outgrowing PC12 cells and substrate NIH/3T3 cells, suggesting that neurofascin expression is also sufficient for neurite induction. Luminex technology for the analysis of protein-protein interactions showed homophilic binding of neurofascin to itself.

Ankyrin-Dependent and -Independent Mechanisms Orchestrate Axonal Compartmentalization of L1 Family Members Neurofascin and L1/Neuron–Glia Cell Adhesion Molecule

“…The idea that NF first travels to the axon generally from the secretory pathway is also suggested by the transient broad axonal localization of NF in very young stage 3 neurons (phase IS1). An early axonal targeting might account for the proposed roles of NF and NrCAM in axon outgrowth and pathfinding in the chick and mouse visual system (Sakurai et al, 2001;Pruss et al, 2004Pruss et al, , 2006Zelina et al, 2005;Williams et al, 2006). After axon outgrowth and synaptogenesis are complete, secretory IS targeting might develop as a mature mechanism to efficiently enrich IS components at the IS later on.…”

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

“…Using Lab-Tek TM 8-well chamber slides (NUNC, Wiesbaden, Germany), 5000 PC12-E2 cells were cultivated on a confluent monolayer of neurofascin-expressing NIH/3T3 in the presence of 100 ng/ml nerve growth factor for 18 -20 h after co-transfection with the indicated expression vectors and an EGFP expression vector for detection of transfected cells, as published previously (8,21). Subsequently, random pictures were taken with a light-amplifying camera (Hamamatsu Photonics) to determine the percentage of process-bearing cells.…”

“…Pharmacological Inhibitors Targeting the FGFR1 Pathway Interfere with Neurofascin-dependent Neurite Outgrowth of Tectal Neurons-We have shown previously that cultured primary tectal neurons extend neurites on substrates of purified neurofascin via homophilic interactions with neurite-bound neurofascin (7,8). The association of neurofascin with FGFR1 suggests a possible involvement of FGFR1 signaling in neurofascin-dependent neurite outgrowth.…”

“…PC12-E2 cells represent a suitable model system to study neurofascin-dependent neurite outgrowth in a co-culture model (7,8). PC12-E2 cells are exposed to a monolayer of NIH/ 3T3 cells for neurite outgrowth assays (Fig.…”

“…Different alternatively spliced neurofascin isoforms are expressed in different cells and at different times of development (6). Embryonal neurofascin NF166 is important for neurite outgrowth and guidance (7,8). Recently, a role for neurofascin NF166 for early processes of inhibitory synaptogenesis at the axon hillock and for the positioning of inhibitory synapses at the axon initial segment has been proven (9,10).…”