Intracellular Trafficking And Secretion Of Cerebral Dopamine Neurotrophic Factor In Neurosecretory Cells

BackgroudCerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved protein that can protect and restore the function of dopaminergic neurons in the rat model of Parkinson's disease (PD), which suggest that CDNF might be beneficial for the treatment of PD. CDNF is widely expressed in neurons in several brain regions including cerebral cortex, hippocampus, substantia nigra, striatum and cerebellum. Human CDNF is glycosylated and secreted from transiently transfected cells, however, the mechanism underlying CDNF secretion is currently unclear.ObjectiveAs a novel neurotrophic factor, fundamental questions about the trafficking and secretion of CDNF remain unanswered. First, how about CDNF secretion, via constitutive or regulated pathway? And which one is the predominant pathway? Second, does glycosylation occur in the CDNF protein synthesis process? Which type, N-linked or O-linked glycosylation? If glycosylation occur, whether it can affect the trafficking and secretion of CDNF protein? Finally, we have known that human CDNF has no pro-sequence. Whether there are any motifs or domains in the CDNF protein can affect its intracellular trafficking and secretion?Methods1. Plasmid constructions2. The secretion of CDNF in transiently transfected PC12 and HEK293 cells3. Immunocytochemical staining of CDNF in both hippocampal neurons and polarized PC12 cells4. Identification of the key amino acids critical for CDNF secretion by use of alanine-scanning mutagenesis5. Co-localization between CDNF and the ER marker-calnexin or the Golgi marker-TGN38 in PC12 cells6. The trafficking of CDNF and its mutants in COS-7 cells7. Co-localization between CDNF and the secretory granule marker-SecllResults1. Localization and secretion of CDNFFirst, we generated a carboxy terminal HA tagged eukaryotic expression construct pcDNA3.1-HA-hCDNF. Then, through co-immunoprecipitation assay and immunocytochemical staining, we confirmed that CDNF is dominantly secreted by the regulated secretion pathway and that CDNF is observed in the soma and processes with a punctate dot staining pattern in both polarized PC12 cells and neurons.2. Glycosylation of CDNF does not affect its secretionWe constructed the N-linked glycosylation and O-linked glycosylation mutant of CDNF:N57A (made a substitution of asparagine for alanine at the position 57) and T181A (made a substitution of threonine for alanine at the position 181). By Western Blot, we found that, when Wt-CDNF and the mutant N57A were transfected into HEK293 cells respectively, the secreted CDNF amount in the N57A medium showed no difference compared with that of Wt-CDNF. When Wt-CDNF and the N57A mutant were respectively transfected into PC12 cells, an established neuronal cell line with both constitutive and regulated secretory pathways, we found that there was no difference in the glycosylated upper bands between Wt-CDNF and N57A mutant in PC12 cell lysate and medium, which suggested that the O-linked but not the N-linked glycosylation of CDNF might occur in PC12 cells. We constructed the T181A mutant. The results showed that the upper glycosylated band was abolished in the cell lysate and conditioned medium of PC12 cells transfected with the T181A mutant, and that the O-linked glycosylation did not affect both the constitutive and regulated secretion of CDNF.3. Key subdomains regulating the secretion of CDNFWe constructed a series of CDNF mutants by inserting a proline into respective a-helices to destabilize the corresponding a-helix structure because proline is a-helix breaker in terms of its hydrogen bonding capability and the bulkiness of its side chain near the backbone. Wt-CDNF and the mutants were transfected into PC12 cells. In M1, which was inserted the proline in the first a-helix, though the protein expression level in cell lysate was normal both constitutive and regulated secretion were significantly reduced. In M7, which was inserted a proline in the seventh a-helix, its constitutive secretion was similar to that of Wt, however the regulated secretion was decreased. Further, we determined that compared with Wt or M7, transfection of M1 leads to a significantly greater distribution in the cell body and decreased distribution in the distal regions of processes. However, the proline insertion in the helix might lead to the misfolding of the whole protein which causes the decreased secretion. To exclude this possibility, alanine-scanning mutagenesis was used to identify the key amino acids in helix-1 critical for CDNF secretion as alanine is compatible with native protein folding. A set of mutants that respective residues in helix-1 were mutated to alanine in combination of four residues were made, and the result suggests that the residues 41-48 in helix-1 are critical for the secretion of CDNF.4. Identification of Trafficking Deficit for Aberrant SecretionM1 mutant was found to co-localize well with calnexin in a typical reticular ER pattern, with significantly greater co-localization percentage than that of Wt-CDNF or M7. Further, we found that M1 was co-localized significantly less with TGN compared with Wt and M7.To further confirm that M1 protein is blocked in the ER and can not be transported from ER to Golgi in the cell, we took advantage of a temperature-sensitive the vesicular stomatitis virus glycoprotein (ts-045-G mutant) (VSVG) tagged with spectral variants of green fluorescent protein (pEGFP-VSVG). Wt-CDNF and M7 partially accumulated in the Golgi when temperature shifts from 39.5℃to 20℃. However, M1 mutant did not accumulate in the Golgi at 20℃, which is different from Wt, M7 and VSVG.5. Defect of M7 in sorting to regulated secretory pathwayWe examined the co-localization of M7 with Secll, an established marker of secretory granules, the result showed that M1 and M7 were significantly less co-localized with Secll compared with Wt-CDNF.ConclusionWe found that CDNF could be secreted primarily via the regulated secretion pathway in PC12 cells. The glycosylation of CDNF is not necessary for its secretion. Moreover, we identified two key subdomains in CDNF which are important for its intracellular localization and secretion. Disrupting helix-1 of CDNF significantly reduces its constitutive and regulated secretion and the helix-1 mutant is retained in endoplasmic reticulum. While helix-7 mutation only decreases CDNF regulated secretion and has no effect on its constitutive secretion, which is further supported by the reduction in co-localization of helix-7 mutant with secretory granules.