Calbindin-D28K expression induced by glial cell line-derived neurotrophic factor in substantia nigra neurons dependent on PI3K/Akt/NF-kappaB 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.

The involvement of NF-kappaB 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 Parkinson's 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.

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.

Integrin beta1 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 alpha (GFRalpha) 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 alpha and beta 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 beta1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin beta1 could form a complex with GFRalphal. The simulation of molecular modeling showed that four H-bonds were formed between integrin beta1 and GFRalpha. These data indicate that integrin beta1 is involved in the transmembrane signaling of GDNF and suggest that integrin beta1 may be an alternative signaling receptor for GDNF.

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

AIM: 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.