Preliminary Studies On Cpne5 Gene Function During The Brain Development

Cpne5 is one of the ubiquitous Ca2+ dependent phospholipids binding proteins that are highly conserved in the evolution and exist in many organisms,from simple livings to complicated mammalians.Eight members in mammalian copine family have been found and named CPNE1-CPNE8 respectively. The Conserved expression and ubiquitous distribution indicate that CPNEs may have important biological roles .Now nothing about Cpne5 has been reported by other groups in the world.Here we show the studies on preparation of polyclonal antibody against Cpne5,Cpne5 expression and possible biological functions.Human CPNE5 is first isolated from fetal brain.It is located on human chromosome 6 of locus p21.1,The mRNA of Human CPNE5,3897bp in full length,contains a 1782bp open reading frame that encodes a protein of 593 amino acids. Mouse Cpne5,4420bp in full length,contains 20 exons and mapped on 17A3.3,also encodes a protein of 593 amino acids.The identity between human Cpne5 and mouse Cpne5 protein is up to 98%.To investigate the expression of Cpne5,we prepared rabbit polyclonal antibody against Cpne5 protein.The results of immunofluorescence and western blot showed that the homemade antibody against Cpne5 Could recognize Cpne5 specificly and Could be used for next experiments.RT-PCR results with mouse fetal brain tissues showed that Cpne5 mRNA was expressed as early as embryonic day 9.5d(E9.5d).The expression level was gradually increased during the gestation stage.Western blot analysis showed that Cpne5 was expressed in the main organ except brain in adult mouse.However,it can be detected in the neonatal mouse brain born wihtin 12 hours. This demonstrates that Cpne5 may play important role in the brain development.To further investigate the distribution and localization of Cpne5 in the embryonic brain,we detected Cpne5 mRNA and protein in the fetal brain by hybridization in situ and immunohistochemistry respectively.The results showed that Cpne5 was mainly expressed in the ventricular zone (VZ). Further investigation demonstrated that Cpne5 was expressed both in the neural progenitor cells and newborn neurons in the ventricular zone.This indicates that Cpne5 might play an important role in neurogenesis of the ventricular zone.To confirm our presumption, we established in utero electroporation model.With the shRNA espression vector targeting Cpne5 mRNA,we knocked down Cpne5 expression in the neurocytes in the ventricular zone (VZ),also the human GFP-CPNE5 expression vector was transfected into the neurocytes in the ventricular zone (VZ).Frozen section analysis showed that inhibiting Cpne5 expression can remarkably increase the number of neuron migrating to the cortex surface,and overexpressed CPNE5 protein can remarkably decrease the number of neuron migrating to the cortex surface.This indicates that Cpne5 works as a negative factor in the radial neuronal migration.Bioinformatic analysis showed that Cpne5 is a Ca2+ dependent phospholipid binding protein.Calcium ion plays important role in the neurogenesis.So we performed a calcium ion binding experiment . The results showed that both endogenous and exogenous expressed Cpne5 have the ability to bind Ca2+ and overexpressed CPNE5 protein can remarkably decrease Ca2+ concentration in HEK293 cells . This suggests that Cpne5 might regulate neuronal migration by binding calcium ion.To further identify Cpne5 roles in brain development,we have established Cpne5 knock-out mouse model.Maybe due to the compensation of undeleted allele,the heterozygotes have no obvious phenotype.The homozygotes are being prepared.In summary,we prepared Cpne5 polyclonal antibody and confirmed the role of Cpne5 in the brain development by examining its expression profiling;By knocking down Cpne5 and overexpressing CPNE5 in VZ cells,we found it functions in the neuronal migration; Cpne5 knock-out mouse model is supposed to help further discuss Cpne5 gene function.