Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis
Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). In addition, inflammation is tightly linked to iron metabolism dysregulation. However, it is not clear whether the brain inflammation-induced iron metabolism dysregulation contributes to the NDs pathogenesis. Herein, we demonstrate that the expression of the systemic iron regulatory hormone, hepcidin, is induced by lipopolysaccharide (LPS) through the IL-6/STAT3 pathway in the cortex and hippocampus. In this paradigm, activated glial cells are the source of IL-6, which was essential in the iron overload-activated apoptosis of neurons. Disrupting astrocyte hepcidin expression prevented the apoptosis of neurons, which were able to maintain levels of FPN1 adequate to avoid iron accumulation. Together, our data are consistent with a model whereby inflammation initiates an intercellular signaling cascade in which activated microglia, through IL-6 signaling, stimulate astrocytes to release hepcidin which, in turn, signals to neurons, via hepcidin, to prevent their iron release. Such a pathway is relevant to NDs in that it links inflammation, microglia and astrocytes to neuronal damage.The NeuN/TUNEL assay was utilized to elucidate the effect of neuroinflammation induced by LPS on neurons in the cortex and hippocampus. The number of NeuN/TUNEL-positive cells in both regions (Figures 1a and b) was significantly increased following intracerebroventricular (ICV) LPS injection.Oxidative stress is one of several factors that can cause neuronal injury. ROS damage membranes, proteins and nucleic acids, ultimately leading to both apoptosis and necrosis. To study whether increased levels of oxidative stress are responsible for the LPS-induced apoptosis observed in the cortex and hippocampus, we examined the levels of ROS, superoxide dismutase (SOD) and malondialdehyde (MDA) after LPS treatment. Compared to the control groups, the levels of ROS and MDA (Figures 1c and d) increased significantly, while SOD levels (Figure 1e) declined markedly in the LPS groups in the two brain regions.Alpha-lipoic acid (αLA) is a compound with strong antioxidant properties. It readily passes across the blood brain barrier and can provide considerable defense against tissue damage induced by free radicals. We examined the number of NeuN/TUNEL-positive cells and the levels of ROS, MDA and SOD after αLA and LPS treatment and found that αLA treatment can significantly reduce the number of NeuN/TUNEL-positive cells (Figures 1a and b) and rescue the changes in ROS, MDA and SOD levels (Figures 1c–e) in LPS-treated mice. This indicates that a high level of oxidative stress likely contributes to the activation of neuron apoptosis after LPS treatment.All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee of Hebei Science and Technical Bureau in PRC. All animals were housed in pairs in stainless steel cages at 21±2 °C and provided free access to food and water. Rooms were humidity controlled room with a 12 h light/dark cycle.Normal male Balb/C mice, weighing 25 g, were purchased from HEB LAC (Hebei Normal University, Shijiazhuang, China). After mice were allowed to adapt to their living conditions for 3 days, 5 μl LPS (2.5 μg/μl, dissolved in sterile 0.9% saline) or vehicle (sterile 0.9% saline) was infused into the right LCV (0.5 mm posterior, 1.0 mm lateral and 2.0 mm ventral to bregma) as previously described.13, 50 The animals were anesthetized (pentobarbital sodium, 40 mg/kg body weight) and then killed for TUNEL and IF, 24 h after LPS injection. The animals were killed for hepcidin mRNA detection 0, 3, 6, 12 or 24 h after LPS injection.αLA (50 mg; Sigma, St. Louis, MO, USA) was weighed and resuspended in 0.35 ml 2 N NaOH. Sterile water (4.5 ml) was added and titrated with approximately 0.05 ml 10 N HCl or until αLA slightly precipitated. The pH was adjusted to 7.2–7.4. The solution was filter sterilized through a 0.2 μm low protein binding syringe filter and frozen in aliquots until used. αLA (100 mg/kg) was administered intraperitoneally once per day for 2 days. LPS was injected 3 h after the final administration of αLA. The animals were anesthetized and then killed for TUNEL and oxidative stress assays 24 h after LPS injection.Male mice with preferential knockdown of hepcidin in astrocytes were generated by Cyagen Biosciences Inc. (Guang Zhou, China). The vector pRP.ExSi-GFAP-shhepcidin-1 (GFAP promoter – human pre-miR30a flanking sequence – sense–loop–antisense (sense strand: 5′-GCAGACAUUGCGAUACCAATT-3′ antisense strand: 5′-UUGGUAUCGCAAUGUCUGCTT-3′) – human pre-miR30a 3′ flanking sequence – bGH polyA) was microinjected into a fertilized egg. Recombinant embryonic stem cells were injected into FVB blastocysts to produce chimeras, which were then crossed to FVB mice to produce heterozygous mice for preferential knockdown of hepcidin in astrocytes (GFAP-shhepcidin). The right LCVs of GFAP-shhepcidin mice were infused with 5 μl LPS (2.5 μg/μl, dissolved in sterile 0.9% saline) or vehicle (sterile 0.9% saline). The animals were anesthetized and then killed for IF assays 24 h after LPS injection.The genotype of GFAP-shhepcidin mice was determined using primer-specific PCR. The primer sequences were as follows: Transgene PCR primer forward 5′-AGCTTTATTGCGGTAGTTTATCACA-3′ and reverse 5′-AAAGTAGCCCCTTGAATTCCGA-3′. The PCR products were resolved by agarose gel electrophoresis. Transgenic mice were identified by the presence of a 457 bp-length PCR product.This work was supported by National Natural Sciences Foundation of China (31471035, 30871260) and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SKLN-201405).Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)Edited by A VerkhrtaskyThe authors declare no conflict of interest.