| BackgroundGlial cell line-derived neurotrophic factor (GDNF) could promote the survival, neurite outgrowth and differentiation of distinct populations of central and peripheral neurons, especially the midbrain dopaminergic neurons and spinal motoneurons, and have been expected as therapeutic agents for neurodegenerative diseases. Many studies in animal models of Parkinson’s disease (PD) and some studies in PD patients show that GDNF delivered to the striatum or the substantia nigra can rescue previously damaged dopaminergic neurons and promote recovery of the motor function. GDNF signals through a multicomponent receptor complex comprising the transmembrane RET tyrosine kinase and a member of a family of glycosylphosphatidylinositol (GPI)-anchored cell surface protein, the GFRα. GDNF mediated RET activation stimulates multiple intracellular signaling pathways including RAS/ERK1/2and PI3K/Akt that promote neuronal survival, migration and neurite outgrowth.Like other neurotrophic factors, GDNF protein is first synthesized in the form of proGDNF and then converted to its mature form during the intracellular trafficking and secretion process. The secretion of GDNF can be either constitutive or regulated by physiological stimuli. GDNF has an alternative spliced isoform GDNFA78with26amino acids deletion in the pro-domain, which has been previously reported to lead to impaired GDNF secretion, suggesting pro-domain may play important role in regulating this process. However, the detailed mechanism driving GDNF secretion is still unknown.Sorting protein-related receptor with A-type repeats (SorLA) is a250-kDa type-1membrane glycoprotein mainly expressed in neurons in the brain. It is a member of a family of five mammalian proteins that share structural similarity with the vacuolar protein sorting10protein (Vps10p), a sorting receptor in Yeast that transports carboxypeptidase Y from the Golgi to the vacuole. The major pool of SorLA was found in late Golgi compartments, with minor expressing on the cell surface. Previous study indicated that SorLA could interact with GDNF. However, the subsequent biological function of this interaction is still not clear, which needs to investigate further.Objective1) To confirm the interaction between SorLA and GDNF2) To seek the key protein that regulates GDNF secretion:investigating SorLA mediation of GDNF secretion further3) To explore the molecular mechanism of this regulationMethodsImmunoprecipitation, western bolt to detect the association of SorLA with GDNFGDNFHA and SorLA were co-transfected into cells by electroporation.48hrs after transfection, lysates were collected and analyzed by immunoprecipitation and western blot. For immunoprecipitation experiments, lysates were collected and incubated with polyclonal HA antibodies or Rabbit anti-Flag antibodies followed by incubation with protein A-Sepharose beads. For the precipitation experiments under acidic condition, the cell lysates were collected in the lysis buffer with a given pH value adjusted using1M HCl. Western blot was performed by separating proteins via SDS-PAGE, transferring to a polyvinylidene difluofide membrane and immunoblotting with HA11or anti-Flag M2antibodies. The immunoreactive protein bands were detected by enhanced chemiluminescence.Immunofluorescent stain to detect co-localization of GDNF and SorLAHippocampi were dissected from embryonic day18rats,7days later, GDNFHA and FlagSorLA were co-transfected into hippocampal neurons. After24hrs, cells were fixed, permeabilized with0.4%Triton X-100and blocked with10%goat serum. Then, the specimens were incubated with primary antibodies and their respective subtype-specific fluorescent secondary antibodies. The immunostained cells were observed with confocal microscope. GDNF secretion assayGDNFHA and SorLA construct or empty vector were co-transfected into PC12cells.48hrs after transfection, cells were washed three times with Krebs’-Ringer’s-Henseleit (KRH) buffer with the following composition (in mM):125NaCl,4.8KCl,2.6CaCl2,25HEPES,1.2MgSO4,5.6Glucose,1Sodium ascorbate, and1.2KH2PO4, adjusted to pH7.4with NaOH. The conditioned media were collected after2hrs incubation at37℃and used as a measure of constitutive secretion. To determine regulated secretion, cells were washed three times with KRH buffer, followed by10min incubation at37℃in stimulated media [KRH buffer with an increased KCl concentration (56mM) and decreased NaCl concentration (75mM)]. The amount of constitutive secretion was normalized to10min. Cell lysates, constitutive and regulated secretion media were analyzed via immunoblotting with HA11antibodies or ELISA assay.ResultsThe interaction of SorLA with GDNFWe expressed both Flag-tagged SorLA and HA-tagged GDNF in PC12cells. The two proteins were detected by specific antibodies against their respective epitope tags, and SorLA/GDNF association was assessed by immunoprecipitation. FlagSorLA was clearly detected in GDNFHA immunoprecipitates, and GDNFHA in FlagSorLA immunoprecipitates. To exclude the possibility that the GDNF and SorLA interaction was because of protein overexpression, endogenous co-immunoprecipitation was performed. The result indicated endogenous GDNF was detected in SorLA immunoprecipitates from rat brain lysates. Furthermore, when FlagSorLA and GDNFHA were co-transfected into cultured hippocampal neurons and immunostained with Flag-antibodies and HA-antibodies, punctate staining of SorLA was found, and co-localized well with GDNF. These data suggest that SorLA interacts with GDNF and localizes to GDNF containing secretory granules. The role of SorLA in regulating GDNF secretion:SorLA enhances GDNF regulated secretion The major SorLA pool is distributed in the trans-Golgi net (TGN), with minor expressing on cell surface. To investigate whether SorLA affects GDNF function on membrane nor in the intracytoplasm, we detected it separately. We failed to detect the interaction of these two proteins on cell surface by immunoprecipitation. And SorLA overexpressing could not enhance the activation of GDNF-RET signal pathways, which suggested that SorLA might not affect GDNF signal on cell surface.Then, whether SorLA regulates GDNF trafficking inside of cells? We found that overexpressing of SorLA could promote GDNF regulated secretion significantly compared to the empty-vector expressing control, while the constitutive secretion did not show obvious change. To confirm this result, we constructed SorLA siRNA and took a truncated SorLA mutant (SorLA-vpslOp, only containing the vpslOp domain, which could interact with GDNF) as the dominant negative mutant. After expression of SorLA or the normal function of SorLA was reduced or inhibited, the regulated secretion of GDNF decreased dramatically. In all, these data suggested that SorLA controls GDNF regulated secretion.Identification the pro-domain of GDNF as the key interaction domain with SorLAPrevious studies have shown the prodomains in neurotrophic factors play important role in their trafficking and secretion, we try to determine which part in GDNF, prodomain or mature domain, could mediate its interaction with SorLA. We constructed GDNF mutants containing only pro-domain or mature domain. Coimmunoprecipitation experiments were performed between pro or mature domain of GDNF and SorLA-vpslOp. Under neutral pH condition, both pro and mature GDNF domain could interact with SorLA Vps10p domain. Because the process of sorting secretion proteins into distinct exocytotic carriers occurs in the trans-Golgi network, and most SorLA is located in the late Golgi compartment, we guess that protein sorting mediated by SorLA may occur in this place. So we performed additional experiments to determine whether it was feasible for such an interaction to occur under the pH6.4condition, the pH value in the lumen of trans-Golgi network. And we found that the prodomain retained whereas the mature domain lost its binding capacity with SorLA Vps10p domain, which suggests that the prodomain of GDNF is the key domain mediating its interaction with SorLA in the Trans-Golgi network. Next we performed the co-immunoprecipitation study under a more acidic condition, pH5.5, the intraluminal pH of mature secretory granules. The pro-domain of GDNF could still bind with the Vps10p domain of SorLA under pH5.5condition, which suggests that GDNF may not only be targeted to but also retain in the secretory granules with binding to SorLA lumen domain in this compartment. Furthermore, we found that SorLA enhanced the regulated secretion of GDNF pro-domain while had no effect on secretion of mature domain.The specificity of SorLA regulating GDNF secretionWe failed to detect the effect SorLA on GDNF△78secretion by overexpressing SorLA or SorLA siRNA. Then, we detected whether sortilin, another member of vpslOp family, could control GDNF regulation. We found that sortilin could associate with GDNF under a neutral condition, however, this interaction disappeared under acidic conditions with pH6.4and5.5. And overexpressing SorLA could not mediate GDNF secretion. These data suggested that SorLA regulated GDNF secretion specifically and indicated the important role of GDNF pro-domain.Conclusion1) SorLA could interact with GDNF, and locates in GDNF containing vesicles2) SorLA enhances GDNF regulated secretion, while has no effect on GDNF constitutive secretion3) The pro-domain is identified as the key domain interacting with SorLANovelty1) The first time to show that a sorting molecule, SorLA, controls GDNF regulated secretion2) We identified that the prodomain of GDNF is the key interaction region with SorLA3) Our data has general implications for the concept that the prodomain of neurotrophic factors plays important roles in their intracellular trafficking and secretion... |
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