The Effect And Mechanism Of GDNF’s High Expression Induced By Stress In The Early Stage Of Dopaminergic Nerve Cell Damage

Background:Parkinson disease(PD) is a common neurodegenerative disease of human central nervous system. The most important pathological feature of PD is a chronic progressive degeneration and death of dopamine(DA) neurons in pars compacta of substantia nigra of patients and how to retard or reverse that progressive degeneration and death of the DA neurons is one of the focuses of current research on Parkinson disease.Glial cell line-derived neurotrophic factor(GDNF) plays a key role in the survival and damage repair of DA neurons. The damaged neurons are induced by stress to start its own molecular process of repairing being, and then activate the specific signal transduction pathways and the related protective gene expression. Other studies have confirmed that DA neurons can express and secrete GDNF. So, in the early stage of damage, is there a similar protective mechanisms induced by stress in DA neurons and whether GDNF is involved in that stress-induced self-repairing? If it does exist, what’s the mechanism underlying the GDNF high expression induced by stress and whether the GDNF high expression has a protective effect on the damaged DA neurons?Oxidative stress damage is an important reason for the degeneration and death of DA neurons in the substantia nigra of PD patients. 6-OHDA is a classic drug for building the PD animal model and the damage induced by 6-OHDA on DA neurons is mainly an oxidative stress damage. As 6-OHDA is administrated into DA neurons, it leads to a transient high expression of the phosphorylated Akt1(a subtype of protein kinase B) in the substantia nigra of PD model rats in the early stage of damage. Another reports said that in tumor cells, Akt1 can directly phosphorylate eye absent 1(Eya1), a protein phosphatase of Eye absent(Eya)family, to activate it. Eya1 is a transcription ancillary factor with a protein serine/threonine phosphatase activity. It is also shown that the co-transfection of Eya1 and Six2 can enhance the transcriptional activity of Six2. It is reported that the transcription factor sine oculis related homeobox 2(Six2) can bind to the GDNF promoter region and promote the transcription of GDNF during the development of kidney. he results of our pre-experiments showed that there were expression level changes of p-Akt1, p-Six2 and p-Eya1 in the DA neurons with 6-OHDA induced damage. So, in the early stage of 6-OHDA induced DA neurons damage, whether the factors of Akt1, Eya1 and Six2 mediates the stress-induced GDNF high expression?Based on the above questions, we proposed to investigate whether there is a stress-induced GDNF high expression existing in the early stage of 6-OHDA induced DA neurons damage; what is the mechnism of the stress-induced GDNF high expression; and whether the stress-induced GDNF high expression has a protective effect against 6-OHDA induced DA neurons damage. Method:In order to observe whether there is a stress-induced GDNF high expression in the early stage of 6-OHDA induced DA neurons damage, using the stereotactic injection of 6-OHDA into the rat striatum, a 6-OHDA induced DA neurons model of the early stage was built; the postural asymmetry test was used to detect the rat motor ability; the real time polymerse polymerase chain reaction(qPCR) and immunoblotting(IB) tests were used to check the GDNF expression. Meanwhile, 6-OHDA was used to build the damaged DA neuron in vitro models; Flow Cytometry and AO/EB staining were used to observe the apoptotic changes of DA neurons; IB and qPCR were taken to detect the expression changes of GDNF; dual luciferase reporter gene assay were taken to detect the transcriptional activity of GDNF. In order to explore the mechanism of the stress-induced GDNF high expression in the early stage DA neurons damage, firstly, we used IB and immunoprecipitation(IP) methods to observe the expression level changes of phosphorylated Akt1(p-Akt1), p-Eya1 and p-Six2 in DA neurons, in vitro. Secondly, plasmids containing Akt1, Eya1, Six2 or knocked- down Akt1 interference sequence(Akt1-shRNA), Eya1-shRNA, Six2-shRNA were constructed, and the IB and IP tests were used to show the expression levels of GDNF, p-Eya1 and p-Six2 as these factors was overexpressed / knocked down respectively; At last, chromatin immunoprecipitation(Ch IP)was taken by anti- Six2, and then qPCR test was used to reveal whether Six2 could bind to the GDNF promoter region and promote the expression of GDNF.In order to further investigate whether the stress-induced GDNF high expression has a protective effect against 6-OHDA induced DA neurons damage, DA cells were cultured and treated with 6-OHDA; when the 6-OHDA treatment was about 2 h, anti-GDNF was used to against the effects of GDNF; when the 6-OHDA treatment was about 4 h, AO/EB staining / Typan Blue staining and cell counting method were used to observe the effects of anti-GDNF on damaged DA neurons apoptosis induced by 6-OHDA. Results: 1. A stress-induced GDNF high expression in the early stage of 6-OHDA induced DA neurons damageStereotactic injection of 6-OHDA into the rat striatum was taken at different time points. the motor ability of the damaged side of rats in Group 1 d was significantly lower than that of the control group, the motor ability of rats in Group 4 d and 7 d was significantly increased compared with that in Group 1 d and 14d; the GDNF mRNA and protein expression of the rat substantia nigra in Group 1 d were significantly decreased than that of the control group, Group 4 d and 7 d of 6-OHDA treatment were significantly increased compared with that in Group 1 and 14 d; The result of immunofluorescence staining showed that there was a GDNF expression in DA neurons of rats substantia nigra.In vitro results showed that the apoptotic rate of DA neurons increased slowerly in 15 min、30 min and 1 h groups after 6-OHDA treatment, then increased fastly in 6 h and 12 h groups after 6-OHDA treatment; GDNF mRNA levels of DA neurons in Group 15 min and 30 min of 6-OHDA treatment were significantly lower than that of the control group, those levels in Group 1 h, 3 h and 6 h was significantly higher than those in Group 15 min and 30 minm and in Group 12 h; the changes of transcriptional activity of GDNF were consistent with that of GDNF mRNA; GDNF protein expression levels in DA neurons in Group 3 h and 6 h were significantly increased also. 2. Akt1 /Eya1/Six2 signaling pathway regulates the stress-induced GDNF high expression in DA neurons with the early stage of damageDA neurons were cultured and treated with 6-OHDA at different time points. The results showed that p-Akt1 expression levels stress-induced up-regulation in DA neurons in Group 15 min, 30 min and 1 h; p-Eya1 expression levels up-regulated in DA neurons in Group 30 min, 1 h and 3 h; p-Six2 expression levels down-regulated in DA neurons in Group 30 min and 1 h.Further experiments showed that overexpression of Akt1 and then injured with 6-OHDA for 1 h, the expression of GDNF and p-Eya1 increased, and p-Six2 decreased; the results of knockdown Six2 were opposite with that of overexpression of Akt1. Overexpression of Eya1 and then injured with 6-OHDA for 1 h, the expression of GDNF increased, and p-Six2 decreased; the results of knockdown Eya1 were opposite with that of overexpression of Eya1. Overexpression of Six2 and then injured with 6-OHDA for 1 h, the expression of GDNF increased; the results of knockdown Six2 were opposite with that of overexpression of Six2; Ch IP-qPCR test showed that the expression level of GDNF in DA neurons in Group 1 h of 6-OHDA treatment were higher than that in the control group. 3. Stress-induced GDNF high expression has a protective effect against 6-OHDA induced DA neurons damageThe cultured DA neurons were treated with 6-OHDA for 2 h, and then anti-GDNF was used to against the stress-induced GDNF expression, results showed that the apoptosis of DA neurons increased siginificantly. Further results showed that the expression level of GDNF in Six2-overexpression DA neurons increased significantly, and the apoptotic rate of DA neurons in Group Six2-overexpression decreased; meanwhile, stereotactic injection of lentivirus particles carrying Six2 into the rat substantia nigra was taken to build PD models; the results showed that the expression level of GDNF increased significantly and motor ability of rats was improved. Conclusion:In the early stage of 6-OHDA induced damage, Akt1/Eya1/Six2 signaling pathway may promote the stress-induced GDNF high expression and thereby protect damaged DA neurons. This study may provide a theoretical basis of a new treatment target, aiming to reverse the DA neurons degeneration and death, for the early treatment of PD patients.