Expression And Function Of Fasciculation And Elongation Protein Zeta-1 (FEZ1) In Astrocytes

Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear.Objective:The aim of this study was (1) to establish and analyze gene expression profiles of cultured T1As and T2As, determine the possible biological function of these genes expressed in T1As and T2As; (2) to confirm the expression and location of FEZ1 in T1As and T2As. (3) to investigate the expression of FEZ1 in rat developing brain; (4) to evaluate the infuence of FEZ1 on cellular activity of astrocytes through transient transfection; (5) to establish FEZ1 fusion protein expression system in the E.coil and evaluate the influence of ectogenic FEZ1 on neurons.Methods:Part one (1) Based on the differential properties of developmental time-course and cellular adhesions in T1A cell linage and O-2A cell linage, Primary rat T1As and T2As were isolated from postnatal day 1 (P1) rat cortex. (2) A DNA microarray was used to identify gene expression in cultured T1As and T2As. Different gene expression profile of purified T1As and T2As were compared and analyzed.Part two (1) Real-time reverse transcription-polymerase chain reactions (RT-PCR), western blots and immunocytochemistry were performed to demonstrate the expression of FEZ1 in cultured T1As and T2As. (2) The expression of FEZ1 in developing brain from rat postnatal 1day, 3 days, 7 days, 2 weeks, 1 month, 3 months were investigated using immunohistochemistry assays.Part three RT-PCR was used to obtain the full length FEZ1 gene from rat cortex. The recombinant plasmids of pEGFP-N1-FEZ1 and FEZ1 RNAi were transfected into asrocyte respectively, and cell activities were examined by MTT. The recombinant protein of FEZ1 with His tag was expressed in prokaryotic cells and its function on dorsal root ganglia outgrowth was observed.Results:(1) Gene profiling, using the BiostarR-40 genechip array, created a transcriptome database of the expression levels of 4096 genes for T1A and T2A. Of the 4069 analyzed genes, a total of 138 were differentially expressed between T1A and T2A, 60 of which were highly expressed in T1As, and 78 in T2As. Approximately 28% (39 of 138) of the genes with altered expression were unknown genes. The remaining 99 genes were involved in metabolism, growth factors, structural molecules, signal transduction, neurite outgrowth, tumors, migration, cell adhesion and transporter activity. There were 57 up-regulated genes (Ratio>2.0) and 42 down-regulated genes (Ratio<0.5) among the 99 known ones. Four genes were found that, according to previous reports, may participate in neurite outgrowth. The four genes were related to the protein tyrosine Phosphatase receptor-type Z polypeptide 1 (PTPRZ1), the dihydropyrimidinase-like 2 (DPYSL2), the fasciculation and elongation protein zeta 1 (FEZ1) and the growth associated protein 43 (GAP-43). All of them were 3-6 fold more highly expressed in T2As compared with T1As. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes.(2) RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1As and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus.(3) Immunohistochemistry assays were performed to demonstrate the expression of FEZ1 in developping rat brain. FEZ1 is preferentially expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes. The highest expression of FEZ1 during the period examined was observed on P7.(4) The recombinant of pEGFP-N1-FEZ1 and FEZ1 RNAi plasmid was constructed successfully. Transient transfection assays showed that inhibition of FEZ1 expression in astrocytes can decrease the cell growth.(5) The recombinant of pET-28a-FEZ1 plasmid was constructed successfully and the fusion protein can promote axon outgrowth of dorsal root ganglia. Conclusions:(1) The present results provided a novel genomewide information for further investigations of T1A and T2A.(2) It was confirmed for the first time that FEZ1 expressed in both types of astrocytes and upregulated in T2A.(3) In rat developing brain, FEZ1 is expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes.(4) The presence of FEZ1 in astrocytes may affect cell behavior and make influence on neurons.The results contribute to a clearer understanding of the expression and function of FEZ1 in the two types of astrocytes.