The Study Of GDNF Isoform Affects Interacelluar Trafficking And Secretion Of GDNF In Cells

Purpose:Glial cell line-derived neurotrophic factor(GDNF) is the prototypic member of a small family of neurotrophic factors that promote the survival,neurite outgrowth and differentiation of distinct populations of central and peripheral neurons,especially the midbrain dopaminergic neurons and spinal motoneurons.GDNF also plays an important role in synapse formation and plasticity.GDNF is widely distributed in the central nervous system including cortex,hippocampus,cerebellum,striatum, hypothalamus,midbrain and spinal cord.GDNF can trigger RET phosphorylation in cooperativity with GFRa1 and induce intracellular signaling and.The significance of GDNF is underscored by its efficacy in several animal models of Parkinson's disease and motor neuron disease.Hopes have been raised that GDNF could be used as a therapeutic agent to treat several neurodegenerative diseases.Like other neurotrophic factors,GDNF is first synthesized as a precursor of 211 amino acids called proGDNF and then cleaved into a mature form of 134 amino acids,due to a proteolytic consensus sequence located in exonⅡ.A shorter GDNF mRNA transcript in human and rodent has been found,which has a 78 bp deletion at the very end of GDNF exonⅠand results in a 26 amino acids deletion in the pro-region of GDNF.However,the 26 amino acids deletion in the GDNF isoform did not affect the GDNF proteolytic sequence.However,little is known about the function of GDNFΔ78.A growing body of evidence suggests that diverse growth factors such as neurotrophins(NTs),insulin-like growth factor-1(IGF-1),and glial cell line-derived neurotrophic factor(GDNF) can be released via the regulated secretory pathway in neuronal cells,possibly representing a mechanism for preferentially supplying these growth factors to active synapses.Whether the GDNFΔ78 affect GDNF processing,trafficking and secretion is unknown.Since GDNF is widely expressed in central neurons,we conducted an analysis to identify the intracellular localization and secretion of GDNFΔ78 in neuronal cells by fluorescent microscopy imaging and Western blot technique.Method:1.Processing and secretion of GDNFFL and GDNFΔ78 in PC12 cells:In order to monitor intracellular localization,processing,trafficking and secretion of GDNF,we through Western blot analysis of transfected PC12 cell lysates,The GDNF ELISA assay further confirmed the result.To determine whether the different processing with GDNFΔ78 has functional consequences,we assessed in PC12 cells whether GDNF's constitutive and regulated secretion were affected.2.Trafficking of GDNFFL and GDNFΔ78 in neuronal cells:An established neuron-like cell line with a polarized morphology,and primary hippocampo-cortical neuronal cells by use of the C-terminal epitope tag staining. By quantitative fluorescence analysis,For the hippocampo-cortical neuronal cultures,we also co-stained for neuronal markers of dendrites(MAP2) to confirm their identity as neurons and to mark the dendrites3.Identification of aberrant GDNFΔ78 trafficking step:To determine which step in the biosynthetic trafficking pathway leads to the altered intracellular distribution of GDNFΔ78,we assessed co-localization with TGN38,an established marker of trans-Golgi network in the secretory pathway.Result:1.By Western blot analysis of transfected PC12 cell lysates,GDNFFL and GDNFΔ78 were expressed at equal levels and both contained the pro and mature form.The GDNF ELISA assay further confirmed that the GDNF amount in the constitutive and regulated secretion media was significant less in GDNFΔ78 group compared with GDNFFL group.There is no difference in the percentage of cells bearing neurites between GDNFΔ78 and GDNFFL group in PC12 cells.2.By quantitative fluorescence analysis,the GDNFΔ78 was localized more in the cell body as compared with the full length GDNF in both PC12 cells and hippocampo-cortical neurons.3.When GDNFΔ78 or GDNFFL was transfected into differentiated PC12 cells or hippocampo-cortical neurons,significantly more GDNFΔ78 was co-localized with the Golgi apparatus marker-TGN38,as compared with GDNFFL.Discussion.Our studies have identified an intracellular trafficking and secretion deficit of the GDNF isoform(GDNFΔ78),which emphasizes the importance of the prodomain in GDNF as a general mechanism of regulating appropriate biological activity.Thus,the different proportion of GDNF isoforms may lead to vulnerability for some neurodegenerative disease.An important direction for future studies will be to link the cell biological defect in GDNFΔ78 trafficking to in vivo functional deficits in human.