Study Of LRRN3 Expression And Its Role In Cerebellum Postnatal Development Regulation

Mechanism study of neural system development is important in Neurobiology. Clarifying of the mechanism will be contribute to the understanding of advanced neural activity including behavior, language and memory. It also can bring light to the prevention and treatment of many neural system associated disease such as aging related disease, development malformation and nerve injury. The development regulation is a multifactorial process, and gene regulation is much more important. Genes and their products can regulate the development and differentiation of neural system through different signal pathway.LRRN3 is a member of Neuronal Leucine-rich repeat super family, with LRR domain, Ig-like C2-type domain and Fibronectin type-â…¢domain in its protein structure, which were reported to be important during neural system development. Some works showed that LRRN3 mRNA was expressed in nervous system of different fetal stage, it indicates that LRRN3 may play roles in neural system development, but we barely know anything about its mechanism till now. Deeply studying the expression and function of LRRN3 protein in neural system will help us to understand the regulation of neural system development, and give fundamental theory support to the prevention and treatment of many neural system related disease.At the first part of the experiment, we prepared rabbit anti-rat LRRN3 polyclone antibody with genetic technology. After analysis the structure of LRRN3, we chose a C-terminal peptide sequence which has high antigenicity and surface expose, and designed primer according to the ORF of Mal-pET21a(+)-His vector. we got the target sequence by RT-PCR and joined them with vector, then transformed the joined products to JM109 and expressed Mal-LRRN3C-His fusion protein in E. coli BL21 induced by IPTG. Using this purified fusion protein as antigen, we prepared and purified anti-LRRN3 polyclone antibody by immune New Zealand Rabbits. And we detected the potency and specificity of antibody by Elisa, Western Blot and immunohistochemistry.At the second part, we used two methods to investigate the LRRN3 protein expression profile especially its expression in neuron system. Bioinformatics methods including EST search strategy and GEO (Gene Expression Omnibus) search strategy were used to predict the expression profile. Result showed that rat LRRN3 mRNA exist in brain, spine nerve root, heart, liver and kidney of embryo and juvenile and mature animals. Then we detected the expression of rat LRRN3 in vivo by immunohistochemistry and Western Blot. In the results, LRRN3 was mostly expressed in central nervous system, positive signals were existed in cerebra cortex neuron,hippocampus dentate gyrus granular cell and cerebella Purkinje cell. And there were no positive signal in other tissues. All these results indicated there are close relationship between LRRN3 protein and nervous system.In order to further investigate the function of LRRN3 in nervous system development, we examined the effects of anti-LRRN3 on cerebellum postnatal development. Behavior experiment, electron microscopy and immunohistochemistry were used to evaluate the effects of anti-LRRN3. In P21 cerebellum, LRRN3 expression of experiment group was lower than the control. Compared with the control, the experiment group showed weak balance ability, the difference of balance latency between them was significant(P<0.05). Electron microscopy results of the experiment group showed neuronal shrinkage in some Purkinje cells and edema in molecular layer, synapse number was obviously less than that of the control. VGluT1 was expressed in cerebellum of these two groups, the positive molecular layer(ML) grew thicker from P7 to P14 and P21, compared with the control, there was no obvious difference in P7 (P>0.05), while in P14 and P21, the difference was significant (P<0.01). The results indicated that LRRN3 protein play an important role in cerebellum postnatal development, anti-LRRN3 antibody injection could partly neutralize endogenous LRRN3, change the ultrastructure of cerebellum, reduce the synapse formation in molecular layer, decrease the expression of VGluT1 and inhibit the growth of cerebellum cortex and the functional neural circuit formation.