FLRT3, a cell surface molecule containing LRR repeats and a FNIII domain, promotes neurite outgrowth

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Abstract

The mature peripheral nervous system has the ability to survive and to regenerate its axons following axonal injury. After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Using cDNA microarrays, we isolated FLRT3 as one of the up-regulated genes expressed in the distal segment of the sciatic nerve 7 days after transection relative to those of the intact sciatic nerve. FLRT3 is a putative type I transmembrane protein containing 10 leucine-rich repeats, a fibronectin type III domain, and an intracellular tail. The neurons plated on CHO cells expressing FLRT3 extended significantly longer neurites than those plated on wild-type CHO cells, demonstrating that FLRT3 promotes neurite outgrowth. FLRT3 mRNA was especially abundant in the basal ganglia, the granular layer of cerebellum, and the hippocampus, except the CA1 region in the adult rat brain. Thus, FLRT3 may contribute to regeneration following axonal injury.

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Materials and methods

All procedures involving animals were reviewed and approved by the local Institutional Animal Care and Use Committee.

Construction of the FLRT3 expression vectors. All expression vectors were constructed using the GATEWAY system (Invitrogen). We constructed two destination vectors, pcDNA5FRT/Igκleader/HA/ccdB and pcDNA/ccdB/HA. An oligonucleotide coding for the HA tag, and a GATEWAY Reading Frame Cassette B, fragment were sequentially ligated into the KpnI–EcoRV sites of the pSecTag2/C vector

FLRT3 is localized in the plasma membrane

Previously, we performed a comprehensive analysis of gene expression following injury in the distal stump of the axotomized sciatic nerve using cDNA microarrays [7]. An EST clone, AI227034, was found to be induced 3.9-fold 7 days after axotomy, and was selected for further study. Induction of the gene expression was confirmed by RT-PCR in the previous study. AI227034 had significant overlap with the RIKEN cDNA, AK017456, which has a putative open reading frame. This protein coded by the clone

Discussion

Upon axotomy, Schwann cells in the distal nerve segment undergo extensive changes along with axonal degeneration. Myelin sheaths are dissoluted and subsequently absorbed by the surrounding cellular environment, a process called Wallerian degeneration. During dissolution and absorption of myelin sheaths by macrophages, the remaining Schwann cells divide and align longitudinally within basal lamina tubes while extending slender cytoplasmic processes, which form the cellular strands called the

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Abbreviations: EST, expressed sequence tag; N-CAM, neural cell adhesion molecule; FNIII, fibronectin type III.

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