IL-1βand TNF-αModulation On Iron Metabolism Of Primary Ventral Mesencephalic Neurons

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized symptomatically by resting tremor, rigidity, and bradykinesia. The neuropathological hallmarks of PD include the progressive loss of dopaminergic neurons in the substantia nigra (SN) and the subsequent depletion of dopamine in the striatum. Although the pathogenesis of PD is still unclear up to now, neuroinflammation, abnormal iron deposit, protein aggregation or mitochondria dysfunction related to environmental, heredity factors or aging were reported to be involved in PD pathogenesis. The prominent hallmarks of neuroinflammation is microglia activation and subsequent secretion of pro-inflammatory cytokines such as interleukin 1β(IL-1β) and tumour necrosis factor-a (TNF-a). Elevated cytokines IL-1βand TNF-a has been detected in the SN and striatum of PD patients and animal models. Direct intra-parenchymal injection of TNF-a and IL-1βinduced dopaminergic neurons degeneration; while blockage of the IL-1βor TNF-a receptor attenuate the death of dopaminergic neurons in 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) induced PD models. Although the mechanisms underlying elevated cytokines induced neurotoxicity are not fully elucidated, PD patients showed amplified levels of pro-inflammatory cytokines in the SN, the same region where iron deposit occured. The relationship between neuroinflammation and iron accumulation becomes compelling. Using fluorometric assays, immunofluorescence, enzyme-linked immunoabsorbent assay, laser confocal scanning microscopy and other methods, the present study aimed to investigate whether and how the pro-inflammatory cytokines IL-1βand TNF-a released by activated microglia, can affect the iron metabolism of ventral mesencephalic (VM) neurons. The results were as follows:1. Iron influx was increased (P<0.01) and iron efflux was decreased (P<0.01) in primary VM neurons with 10 ng/ml IL-1βor TNF-a treatment for 24 h.2. Divalent metal transporter 1 with the iron response element (DMT1+IRE) was up-regulated on both protein and mRNA levels (P<0.05), and ferroportinl (FPN1) was down-regulated on both protein and mRNA levels (P<0.05) in primary VM neurons with 10 ng/ml IL-1βor TNF-a treatment for 24 h.3. Iron regulatory protein (IRP) 1 protein level was up-regulated in primary VM neurons with 10 ng/ml IL-1(3 or TNF-a treatment for 24 h (P<0.05).4. Reactive oxide species (ROS) and nitric oxide (NO) generation was enhanced in primary VM neurons with 10 ng/ml IL-1βor TNF-a treatment for 24 h (P<0.01).5. IL-1βand TNF-a induced IRP1 activation was fully abolished in primary VM neurons by pretreatment with 0.5 mmol/L N-acetyl-1-cysteine (NAC) and 1 mmol/L Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) due to the complete blockage ROS and NO generation.6. Hepcidin mRNA expression was increased in primary VM neurons with 10 ng/ml IL-1βor TNF-a treatment for 24 h (P<0.05).7. IL-1βand TNF-a release was significantly increased in primary microglia with 800 ng/ml lipopolysaccharide (LPS) treatment for 48 h (P<0.01), and this process was enhanced by 100μm/L ferric ammonium citrate (FAC) (P<0.05), but attenuated by 100μm/L desferrioxamine mesylate (DFO) (P<0.05).The above results suggest that IL-1βand TNF-a can induce activation of IRP1, thus up-regulated DMT1+IRE expression and down-regulated FPN1 expression, which are responsible for the increased iron influx and decreased iron efflux of VM neurons. ROS and NO may be responsible for the activation of IRP1; which was fully abolished by co-administration of radical scavenger NAC and nitric oxide synthase inhibitor L-NAME. In addition to IRP1, hepcidin also participate in down-regulation of FPN1. And microglia can be activated by LPS, resulting in abundant IL-1(3 and TNF-a secretion. This process is enhanced by iron repletion and attenuated by iron depletion. Our findings provide evidence that microglia play an important role in neuron iron homoeostasis by secreting IL-1(3 and TNF-a. Oxidative stress and NO induced by IL-1β and TNF-a activated IRP1, which regulated expression of DMTl+IRE and FPN1, thus leading to neuron iron load and even demise. Our findings provide powerful evidence that the cooperative effect of neuroinflammation and iron metabolism may enhance dopaminergic neurons demise in PD and further support that anti-inflammation could be valuable therapeutic approaches in PD.