Parkinsons disease is characterized by the irreversible and selective loss of nigrostriatal dopaminergic neurons.Glial cell line-derived neurotrophic factor (GDNF) is an essential survival and maintenance factor for adult dopaminergic neurons,and is considered a promising neuroprotective candklate for Parkinsons disease.However,the signaling mechanisms underlying this protective effect remain unclear.MicroRNAs (miRNAs) are endogenous,non-protein-coding regulatory RNA molecules that regulate the expression and translation of target genes.miRNAs are involved in a number of neurodegenerative diseases.Both miRNAs and GDNF affect dopaminergic neuronal processes,but the molecular crosstalk between these molecules is not fully elucidated.To understand the molecular basis of the responses of injured dopaminergic neurons to GDNF,it is important to determine if miRNAs expression or function is modified by GDNF.The present study evaluated whether GDNF modulates miRNAs expression.We used microarray analysis and real-time polymerase chain reaction (RT-PCR) to determine miRNAs expression in 6-hydroxydopamine (6-OHDA)-injured MN9D cells treated with GDNF for 30 minutes,1 hour,or 3 hours.We found that at 30 minutes,1 hour,and 3 hours of GDNF treatment,27,46,and 70 miRNAs,respectively,were differentially expressed and,among them,26% (7 of 27),67% (31 of 46),and 100% (70 of 70) were up-regulated,while the remaining were down-regulated.As GDNF treatment time increased,so did the number of differentially expressed miRNAs; the number of up-regulated miRNAs increased,and the number of down-regulated miRNAs decreased.However,we noted that miRNA expression profiles changed quickly after a relatively short GDNF treatment,and few miRNAs showed the same direction of change at all three time points.This reflects the complexity of miRNA regulatory networks.Using Cluster 3.0,we found that miRNAs with similar expression profiles clustered together in four patterns.Bioinformatics analysis of deregulated miRNAs after GDNF treatment predicted that these miRNAs may be predominantly associated with biological processes such as signal transductJon,metabolism,development,intracellular signaling cascades,anatomical structure development,and multicellular organismal development.These miRNAs may also be involved in pathways including axon guidance,cancer,neuraactive ligand receptor interactions,neurotrophin signaling,cytokine-cytokine receptor interactions,endocytosis,and WNT signaling,at all three time points.Bioinformatics algorithms have played a key rale in the discovery of miRNAs,in target gene prediction,and in developing the miRNAome.Although a variety of miRNA target prediction algorithms are available,their results are often inconsistent.To reduce the likelihood of false positives and to increase prediction accuracy,we selected target genes of differentially expressed miRNAs predicted simultaneously by at least three programs.However,we still identified thousands of target genes.Therefore,we selected a single time point (1 hour) to identify and confirm target genes that are truly related to miRNA regulatory function.We simultaneously detected miRNA and mRNA expression profiles from the same samples at 1 hour and compared their expression profiles.Putative miRNA target genes that were consistent with mRNA expression results were chosen for further analysis.We found that the number of target genes of deregulated miRNAs were dramatically reduced using this strategy.Among the target genes predicted by bioinformatics algorithms,only a small fraction of genes is actually involved in biological processes.This method did not reduce the target miRNA list,but it did narrow the list of predictive target genes,indicating that miR-532-5p,miR-188-5p,miR-434-3p,miR-695,miR-340-3p,miR-761,miR-691,miR-342-5p,and miR-697 may be the most important modulators after 1-hour GDNF treatment.We found that these target genes were involved in biological processes such as regulation of cellular component organization and biogenesis,signal transduction,development,transcription from the RNA polymerase II promoter,and metabolic processes.These genes were also involved in biological pathways,including mTOR,neurotrophin,insulin and JAK/STAT signaling,as well as neuraactive ligand receptor interactions,cytokine-cytokine receptor interactions,endocytosis,and the complement and coagulation cascades.These results demonstrate that the identified biological processes and pathways were different from the results derived from miRNA alone.This might be due to the limitations of computational approaches to predict miRNA targets.Collectively,these results provide evidence for the capacity of GDNF to influence miRNAs expression,and show that miRNAs modulated by GDNF represent a novel genetic pathway controlled by neurotrophic factors that could affect GDNF-dependent biological processes.