| Parkinson’s disease (PD) is a neurodegenerative disorder characterized in its late phase by the sustained loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinically PD is characterized by bradykinesia, rigidity, rest tremor and postural instability. The mechanisms underlying PD pathogenesis have not been revealed yet. Multiple factors might be involved, such as genetic mutation, environmental factors, aging, et al. Two of pathological features in PD are oxidative stress and nigral iron deposit. Excessive iron enhances the production of oxygen radicals and thus is associated with oxidative damage and eventually neuronal degeneration in this region. In animal models and PD patients, selective deposition of iron in the SN has been observed, while no iron content increases in other brain regions. Application of iron chelator could exert significant neuroprotective effects in PD animal models. In early stage of PD patients, increased SN iron content could be observed using MRI, and the level of iron was associatd with the disease progressions. So far most of the studies are concerned with iron accumulation in melanized dopaminergic neurons. Little is known about the contribution of astrocytes to this iron accumulation under oxidative circumstances.Astrocytes, the major glial cell type of the central nervous system, constitute up to20%-50%of the brain volume, and have multiple functions, including cellular support during CNS development, ion homeostasis, uptake of neurotransmitters, contribution to the CNS immune system and neuromodulation. Astrocytes participate in the formation of blood-brain barrier (BBB) and are generally accepted as principle contributor for the uptake of a variety of nutrients such as transition metals crossing the BBB. Brain divalent metal transporterl (DMT1) was mainly expressed in astrocytes’end feet around the capillary, indicating a major role of astrocytes in brain iron uptake. These cells have a strong ability to accumulate iron by storage in ferritin and transfer iron back into the blood through the iron transporter ferroportinl (FPN1). Astrocytes have a high tolerance for iron. Iron overloading can trigger neuronal cell and microglia death, however, astrocyte proliferation. Thus, astrocytes play an important role in regulating brain iron homeostasis and protecting other brain cells from iron-mediated oxidative stress. In the present study, using primary cultured astrocytes and ventral mesencephalon (VM) neurons as cell models, we used laser confocal scanning, RNA interference technique, western blot, multi-immunofluorescence labeling, real-time PCR and other methods to elucidate that whether and how iron homeostasis was altered in astrocytes responded to6-OHDA, and the effect of neurotrophic factors (NTFs), such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived growth factor (GDNF) on the iron influx in primary cultured VM neurons, as well as the underlying mechanisms. The results were as follows:1. There was a significant decrease in the fluorescence intensity in primary cultured astrocytes after10μM and100μM6-OHDA treatment for24h compared with control when perfused with1mM ferrous iron for0.5h. When cells were perfused with1mM DFO, a greater reverse in6-OHDA-treated groups was observed. These results indicated that6-OHDA could enhance iron transportation rate in primary cultured astrocytes.2. In accordance with these iron traffic modulations, DMT1+IRE protein levels in10μM6-OHDA treated astrocytes were up-regulated to1.51folds compared with control. A more dramatic elevation of DMT1+IRE in100μM6-OHDA group was observed as1.95-fold. The expression of FPN1, the only channel for iron export, was up-regulated about1.40and1.29-fold above control level. Double immunofluorescence labeling also showed that6-OHDA increased DMT1+IRE and FPN1expressions in astrocytes after6-OHDA treatment for24h. DMT1+IRE was observed in nucleus and cytoplasm, and FPN1predominantly located in the soma and processes of rat astrocytes. Similar to what we found on the protein levels, real-time PCR analysis showed DMT1+IRE mRNA levels in10μM and100μM6-OHDA treated groups were up-regulated about1.9and3-fold compared with control, and FPN1mRNA levels were increased to1.8and2.7-fold compared with control. The difference between10μM and100μM6-OHDA groups was significant.3. Iron regulatory protein1(IRP1) showed a dynamic regulation with10μM and100μM6-OHDA treatment in primary cultured astrocytes. Up-regulation of IRP1protein levels to about1.52and1.31-fold was observed at12h in primary cultured astrocytes after6-OHDA incubation. However, this response was transient, and a declined expression of IRP1even31%and21%below basal levels was observed at24h of6-OHDA treatment.4.6-OHDA treatment for0.5h could induce activation of nuclear factor kappaB (NFkB) p65. Pre-incubation with IκBα activation inhibitor BAY11-7082(5μM) for0.5h could fully block6-OHDA induced NF-κB p65phosphorylation.6-OHDA induced translocation of phospho-NF-κB p65could observe at24h. Pre-incubation with BAY11-7082for0.5h before treatment with6-OHDA (10μM and100μM) for24h could inhibit6-OHDA induced DMT1+IRE up-regulation, indicating the involvement of NFκB p65activation in this process.5. BDNF and GDNF mRNA expression quickly reached the peak in primary cultured astrocytes with10μM,100μM6-OHDA treatments for6h. The BDNF mRNA levels in10μM6-OHDA group were3.4-fold,1.9-fold of control for6h,12h incubation respectively. In100μM group the mRNA levels were2.4-fold,1.6-fold respectively. At24h, BDNF mRNA levels in6-OHDA treated cultures were back to basal levels. Similar tendency was found on GDNF mRNA levels.10μM,100μM6-OHDA treatment induced significant elevations of GDNF mRNA levels at6h (3.3,5.1-fold for10μM and100μM, respectively), and more sustained elevations over time (1.5,1.9-fold at12h, and1.8,2.1-fold at24h)6. In10μM6-OHDA-treated VM neurons for24h, increased ferrous iron influx was observed. Pre-incubation of BDNF (10ng/ml) or GDNF (10ng/ml) for4h prior to6-OHDA (10μM) treatment for anther24h could block6-OHDA enhanced ferrous iron influx in primary cultured VM neurons.7. In10μM6-OHDA-treated VM neurons, DMT1+IRE protein level was up-regulated to1.5-fold compared with control. Sole BDNF or GDNF (10ng/ml) treatment for24h could suppress DMT1+IRE protein levels to nearly26%or21%below the control, respectively. Pretreatment with BDNF/GDNF could fully block6-OHDA induced DMT1+IRE up-regulation. Real-time PCR showed BDNF/GDNF incubation down-regulated DMT1+IRE mRNA levels to50%or40%to the basal level, respectively, and pre-incubation of BDNF/GDNF could fully abolish6-OHDA induced DMT1+IRE mRNA up-regulation.8. Both10ng/ml BDNF and GDNF treatment for24h could down-regulate the basal level of IRP1expression about19%or17%. IRP1protein levels in10μM6-OHDA treated VM neurons were up-regulated and the elevated levels of IRP1could be fully abolished by pretreatment with BDNF/GDNF for4h prior to6-OHDA incubation. With RNA interference of IRP1, the down-regulation of DMT1+IRE induced by BDNF/GDNF was not further observed in MES23.5dopaminergic neurons.9. Significant increased levels of phosphorylated ERK1/2within0.5h after BDNF administration was observed, and the phospho-ERK level was gradually returned but still above the basal level at4h. The expression of phosphorylated Akt was similar to that of phosphorylated ERK1/2. Maximum activation of ERK1/2was achieved also within0.5h after GDNF treatment, however, a weaker response compared to that in cell with BDNF exposure. GDNF also induced Akt phosphorylation at0.5h, however, back to basal level at4h. Double immunofluorescence labeling also showed phospho-ERK and phospho-Akt were observed in MAP2positive neurons with BDNF/GDNF treatment for0.5h. Mitogen-activated protein kinase kinase (MEK) inhibitor PD98059(5μM) or PI3K inhibitor LY294002(2.5μM) pre-incubation for0.5h could fully abolish BDNF/GDNF induced ERK1/2and Akt phosphorylation, respectively.10. Pretreatment with pharmacological inhibitors, PD98059(5μM), LY294002(2.5μM) for0.5h prior to BDNF/GDNF (10ng/ml) incubation for24h could abolish down-regulation of IRP1and DMT1+IRE on both protein and mRNA levels induced by BDNF/GDNF. Remarkably, pre-treatment with PD98059could even up-regulate the expression of DMT1+IRE mRNA in BDNF/GDNF-treated cells.These results suggested that6-OHDA might promote iron transport rate in astocytes under the condition of oxidative stress, thus to avoid iron deposition in astrocytes and participate in brain iron homeostasis. The functional changes were in consistent with iron transporters DMT1+IRE and FPN1up-regulation. Regulations of IRP1expression and NFκB p65activation might post-transcriptionally and transcriptionally influence DMT1+IRE, FPN1expression. Meanwhile,6-OHDA induced elevations of BDNF/GDNF levels in astrocytes. BDNF/GDNF could down-regulate IRP1expression, attenuate6-OHDA-induced improper up-regulation of IRP1which post-transcriptionally regulate DMT1+IRE expression. Intracellular signaling pathways MEK/ERK, PI3K/Akt might participate in these processes. In this way, BDNF, GDNF protect VM neurons against6-OHDA-induced iron accumulation. This study might provide new information on iron homeostasis modulation of astrocytes under oxidative circumstances occurred in PD. |
