Experimental Study Of EphA5 Inducing The Projection Of Mesocephalic Neural Epithelial Stem Cells To Striatum

Parkinson's disease(PD) is a kind of neural degenerative disease with high prevalence and known as a common and frenquently-occuring disease in old people.The main pathophysiological change is the selective degeneration of midbrain dopaminergic neurons.Nowadays,more and more old people are suffering this disease.The severe influence on health and heavy burden to society motivate the study of pathological process and effective treatment for PD.Over the past few years,gene and environment factors were proved to be related to PD. Thus,scholars all over the world made hypotheses include genetical hypothesis,oxidative stress hypothesis,iron metabolism hypothesis et al.All the factors above are related to selective degeneration of midbrain dopaminergic neurons.Therefore,the abnormality of midbrain dopaminergic neurons and their projection to striatum is the neuroanatomical basis of PD.The recent study shows that axon guidance molecule EphA5 is related to the target growth of midbrain DA neurons. The expression of EphA5 is almost the same as that of tyrosine hydroxylase(TH) in DA neurons of midbrain.EphA5 is a kind of axon guidance molecule which binds with its corresponding ligand may induce the growing of midbrain axons.Based on the ideas above,we designed the following experiment.(1) With the techniques of immunocytochemisty staining,RT-PCR and Western blotting, we examined the expression of EphA5 in neuroepithelial stem cells(NEPs) and the expression of Ephrin-A5 in striatum.(2) In order to analyze whether EphA5 involves in the projection of midbrain DA neurons to striatum.We injected retrograde axon tracer in striatum of new-born rats, then the midbrain DA neurons were examined by immunohistochemistry staining.(3) We cocultured midbrain NEPs from GFP transgenetic mice with normal-rats' striatum in vitro and observed the target growth of NEPs axons.Meanwhile,we examined the effect of striatum extract on promoting axon growth.(4) Further more,using RNAi technique to silent the expression of EphA5,we observed the axon growth of electrobloted NEPs cocultured with striatum explants.Results:(1) After 24 h in vitro culture, the NEPs which were derived from midbrain of embryos adhered on the wall and grew short neurites.After 48 h in culture, the result of immunocytochemisty showed that most of cells were TH positive, and part of them were EphA5 positive.(2) Ephrin-A5 is the ligand of EphA5,so we examined the expression of Ephrin-A5 in striatum.The results showed most of cells in striatum were Ephrin-A5 positive.(3) RT-PCR and Western blotting techniques were performed to detect the expression of mRNA and protein of Ephrin-A5.Results showed that the expression of Ephrin-A5 in experimental group was higher than that of control group(P<0.01).(4) The results of retrograde tracing and immunohistochemistry indicated that most of the labeled neurons were TH positive and part of them were EphA5 positive.(5) After 12 h in vitro coculture, the NEPs were round-shaped without neuritis.However, the cells near striatum explants grew neurites immediately. After 24 h,the cells stretched out short neurites,after 3 d,the soma of adhesive cells demonstrated triangle or polygon and had significant trends of growing towards striatum explants.Further more,NEPs derived from GFP transgenetic mice were cocultured with striatal explants.We observed that the cells with greenfluorence stretched out neutrites toward the explant, the number and the length of neurites toward striatum explants were obviously higher than that in the adverse direction(P<0.01). Meanwhile,with striatum extracts in medium,we found the number and the length of neuritis were both higher than those in normal culture (P＜0.01).(6) The electroblotted NEPs were cocultured with striatum explants, results showed that the trend of growing towards striatum was not significant(P＞0.05).In conclusion,axon guidance molecule EphA5 is positive in midbrain NEPs and the expression of its ligand Ephrin-A5 in striatum is also positive.Axon guidance molecule EphA5 may induce midbrain NEPs to project to striatum.