Dian-Shuai Gao

Integrin β1 is involved in the signaling of glial cell line-derived neurotrophic factor

Glial cell line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the substantia nigra (SN) dopamine (DA) neurons. The transmembrane signaling of GDNF is mediated by a unique receptor system, including the ligand binding receptor GDNF family receptor α (GFRα) and the transmembrane signaling receptor Ret or neural cell adhesion molecule‐140 (NCAM‐140). Here, we found that another transmembrane cell adhesion molecule, integrin, a heterodimer consisting of α and β subunits, also mediates the transmembrane signaling of GDNF. The results showed that the level of phosphorylated Src homology 2 domain containing (Shc), which was associated with the cytoplasmic domain of integrin β1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin β1 could form a complex with GFRαl. The simulation of molecular modeling showed that four H‐bonds were formed between integrin β1 and GFRα. These data indicate that integrin β1 is involved in the transmembrane signaling of GDNF and suggest that integrin β1 may be an alternative signaling receptor for GDNF. J. Comp. Neurol. 509:203–210, 2008.

Involvement of NCAM in the effects of GDNF on the neurite outgrowth in the dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) exerts its biological effects via a multi-component receptor system including the ligand binding receptor--GDNF family receptor-alpha1 (GFRalpha1) and the signaling receptor--RET tyrosine kinase. Recently, the neural cell adhesion molecule (NCAM) has been identified as an alternative signaling receptor for GDNF. The purpose of this study was to investigate whether NCAM could mediate the protective effect of GDNF on injured dopamine (DA) neurons and to determine which cytoplasmic signal molecule associated with NCAM was activated while GDNF performing this effect. The results showed that the phosphorylation of NCAM-associated Fyn was upregulated with GDNF treatment, and this upregulation was inhibited by pre-treatment with the NCAM function-blocking antibody. Moreover, pre-treatment with the antibody could abolish the effect of GDNF on promoting the neurite outgrowth of these DA neurons, except for the effect of GDNF on promoting the expression of tyrosine hydroxylase (TH) in these DA neurons. These results suggest that NCAM is involved in the promotive effect of GDNF on the neurite outgrowth in lesioned DA neurons, but not involved in the promotive effect of GDNF on TH expression in these neurons.

The involvement of NF-κB p65/p52 in the effects of GDNF on DA neurons in early PD rats

Glial cell line-derived neurotrophic factor (GDNF) can exert neuroprotective effects on the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons that are undergoing degeneration in Parkinsons disease (PD). In an attempt to investigate the molecular signaling mechanisms underlying GDNF protection the DA neurons from degeneration, we established early PD rat models in which the DA neurons in SNc were degenerating. Whether the cytoplasmic NF-kappaB signaling pathway was involved in the protection of GDNF on the degenerating DA neurons was examined in the present study. The results showed that the nuclear NF-kappaB p65 levels in the DA neurons increased when GDNF was injected into SNc of early PD rat models. Immunoprecipitation assays showed that the nuclear NF-kappaB p65/p52 complex levels increased after GDNF administration, while the p65/p50 complex levels decreased. These results indicated that GDNF could activate the NF-kappaB signaling pathway in the degenerating DA neurons. And it was the noncanonical NF-kappaB signaling pathway, which contained the NF-kappaB p65/p52 complex that was involved in the effects of GDNF on DA neurons.

Calbindin-D28K expression induced by glial cell line-derived neurotrophic factor in substantia nigra neurons dependent on PI3K/Akt/NF-κB signaling pathway☆

Calbindin-D28K is a calcium-binding protein in neuronal cytoplasm, which has the capability to protect neurons from degeneration. It was reported that glial cell line-derived neurotrophic factor (GDNF) increased calbindin-D28K expression in dopaminergic neurons in vitro. It was observed in our research that GDNF also enhanced the expression of calbindin-D28K in adult rat substantia nigra neurons in vivo. To investigate the intracellular signaling pathways underlying the calbindin-D28K expression induced by GDNF, immunoblot and immunoprecipitation analyses were performed in our present study. Our results showed that injection of GDNF alone into substantia nigra of an adult rat brain increased the calbindin-D28K expression; meanwhile, the phosphorylation level of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) increased. However, the calbindin-D28K expression induced by GDNF was specifically blocked by the inhibitor of phosphatidylinositol 3-kinase (PI3K), but the inhibitor of ERK1/2 did not block the calbindin-D28K expression. Furthermore, GDNF administration also caused the nuclear factor kappaB (NF-kappaB/p65), to translocate from cytoplasm into the nucleus, and the inhibitor of PI3K effectively blocked the translocation. Immunoprecipitation assay results further demonstrated that it was the p65/p52 complex of NF-kappaB, rather than the p65/p50 complex that translocated into the neuronal nucleus. The calbindin-D28K expression induced by GDNF was also inhibited when the NF-kappaB signaling pathway was blocked by Helenalin. These results described a novel mechanism by which the activation of PI3K/Akt-->NF-kappaB (p65/p52) signaling pathway could play a role in the calbindin-D28K expression induced by GDNF.

Calbindin-D28K inhibits apoptosis in dopaminergic neurons by activation of the PI3-kinase-Akt signaling pathway

Calbindin-D28k (CaBP) has a neuroprotective effect on dopaminergic (DA) neurons in several models of Parkinsons disease. We used the DA cell line MN9D to explore the mechanisms underlying CaBP-mediated protection against the neurotoxin 6-hydroxydopamine (6-OHDA) of DA neurons. In MN9D cells that were transfected with the expression vector pcDNA3-CB containing CaBP cDNA, the expression level of CaBP was significantly increased. After treating with 6-OHDA, a significant decrease in the apoptosis rate of the transfected MN9D cells was noted, as well as an obvious increase in the expression of phosphorylation of Akt (p-Akt); however, no significant change in the expression of total Akt or phospho-p100 (p-p100) occurred after this treatment. After treatment with wortmannin, an inhibitor of the PI3-kinase-Akt (PI-3K/Akt) signal pathway, an increase in the expression level of CaBP was observed, but there were no other obvious changes of the experimental index mentioned previously in the groups transfected with pcDNA3-CB. These studies suggest that CaBP has a significant role in protecting DA cells against the apoptosis induced by 6-OHDA--through PI-3K/Akt signaling pathway--where the non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway might have no relevance.

NF-κB p65/p52 plays a role in GDNF up-regulating Bcl-2 and Bcl-w expression in 6-OHDA-induced apoptosis of MN9D cell

Glial-cell-line-derived neurotrophic factor (GDNF) has been shown to protect dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA) toxicity. The mechanism underlying the antiapoptosis role of GDNF still needs further studies. We previously observed that nuclear factor-kappaB (NF-κB) signaling pathway, i.e. p65/p52, mediated the antiapoptosis role of GDNF in MN9D cells. Here, the DA cell line MN9D was used to explore the mechanisms underlying NF-κB p65/p52-mediated protection role of GDNF in DA neurons. The results showed that GDNF pretreatment blocked the apoptotic effects induced by 6-OHDA, with the upregulation of the antiapoptotic protein, Bcl-2 and Bcl-w, as well as the downregulation of the proapoptotic proteins, Bax and Bad. Furthermore, when sip100 plasmids were transfected into MN9D cells to inhibit the expression of p100, which was the precursor of p52, the effects of GDNF on upregulating Bcl-2 and Bcl-w were attenuated. These results indicated that GDNF could protect MN9D cells from apoptosis induced by 6-OHDA via upregulating Bcl-2 and Bcl-w expressions and downregulating Bax and Bad expressions. Moreover, NF-κB p65/p52 signaling mediated the effects of GDNF on Bcl-2 and Bcl-w expressions.

Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells

AbstractAim:To explore the intracellular mechanisms underlying the survival/differentiation effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine (DA) cells.Methods:Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting.Results:GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76±0.75; GDNF group, 18.63±0.95. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: control group, 3.65±0.88 and 2.49±0.42; GDNF group, 6.01±0.43 and 4.89±0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98±0.58. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Akt pathway-inhibited group, 3.79±0.62 and 2.50±0.25, respectively).Conclusion:The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.

Global MicroRNA Expression Profiling Reveals Differential Expression of Target Genes in 6-Hydroxydopamine-injured MN9D Cells

Recent evidence indicates that microRNAs (miRNAs) play a key role in neurodegenerative diseases. However, little is known about how these small RNAs contribute to dopaminergic neuronal apoptosis. Here, we profiled the expression of miRNAs in MN9D cells with and without 6-hydroxydopamine (6-OHDA) treatment by miRCURY™ LNA microRNA arrays. We identified six miRNAs (miR-668-3p, let-7d-3p, miR-3077-3p, miR-665-5p, miR-99b-3p, and miR-323-3p) that were significantly lower and five miRNAs (miR-875, miR-207, miR-425-5p, miR-19b-3p, and miR-338-3p) that were significantly higher after 6-OHDA treatment. Among them, five have been demonstrated to be implicated in neurodegenerative diseases. Consistent with our prediction, the deregulated miRNA’s target mRNAs, such as peroxiredoxin III (Prx III) and Myc, also showed changes in their expression levels. Furthermore, using a dual-luciferase reporter assay, we confirmed that Prx III was a direct target gene of miR-875. Taken together, these findings demonstrate that changes in miRNA expression occur after 6-OHDA treatment and suggest that miRNAs and their predicted targets have a potential role in apoptosis of MN9D cells.

Negative feedback loop between p66Shc and ZEB1 regulates fibrotic EMT response in lung cancer cells

The epithelial-to-mesenchymal transition (EMT) program is crucial for the epithelial cancer progression and fibrotic diseases. Our previous work has demonstrated that p66Shc, a focal adhesion-associated adaptor protein, is frequently downregulated in lung cancers and its depletion promotes metastasis behavior through anoikis resistance. However, mechanism underlying loss of p66Shc and EMT response is not fully understood. Here, we showed that p66Shc deficiency enhanced the expression of ZEB1, the known mesenchymal transcription factor and consequently increased Vimentin, and decreased epithelial markers of E-cadherin and β-catenin. p66Shc depletion also increased cell invasion and migration. In addition, ChIP and luciferase assays showed that these effects were directly mediated by ZEB1 repression of p66Shc promoter. Thus, our findings define a critical role of p66Shc in the suppression of fibrotic EMT response with a negative feedback loop between p66Shc and ZEB1 in lung epithelial cancer cells.

Expressions and possible roles of GDNF receptors in the developing dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) has an essential role in the survival and maturation of the dopaminergic (DA) neurons in the substantia nigra (SN) of mammalian embryonic brain. In addition to Ret, cell adhesion molecules (CAMs) were also proposed to function as transmembrane signaling receptors of GDNF. The present study was to investigate whether these transmembrane receptors of GDNF were correlated with the tyrosine hydroxylase (TH) expression of SN DA neurons during early developmental stage. RT-PCR and Western blot were performed to detect TH expression in SN of perinatal rats at mRNA and protein level respectively; meanwhile, Western blot was performed to detect the expressions of the transmembrane proteins including Ret, neural cell adhesion molecule-140 (NCAM-140), integrin β1 and N-cadherin. The results showed that TH mRNA expression was positively correlated with both Ret and N-cadherin protein, while there was no correlation with NCAM-140 and integrin β1; TH protein expression was correlated with all of these transmembrane molecules. These data suggested that the expression of either TH mRNA or TH protein was subject to the mediation of different transmembrane receptor combinations of GDNF.