Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression

Yukiko Doi,Jianfeng Liang,Akio Suzumura,Hideyuki Takeuchi,Mariko Noda,Yoshifumi Sonobe,Tetsuya Mizuno,Jun Kawanokuchi

doi:10.1074/jbc.m110.169839

Abstract

Glutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals.

Highlights

Son disease, where this cell type is thought to have both neurotoxic and neuroprotective properties [1]
We have shown that microglia activated by the Toll-like receptor 9 ligand CpG attenuate oligomeric amyloid ␤ (A␤) neurotoxicity by producing the antioxidant enzyme heme oxygenase-1 (HO-1) and phagocytosing A␤ [4]
Neurons Exposed to Glutamate Release soluble FKN (sFKN), Promoting Microglial Clearance of Neuronal Debris—The expression of FKN and its receptor CX3CR1 was examined in neurons and microglia

Summary

EXPERIMENTAL PROCEDURES

Reagents—L-Glutamate, goat IgG, and LPS were purchased from Sigma (St. Louis, MO). Anti-mouse MFG-E8 antibody and JNK peptide inhibitor (L-JNKI) were purchased from MBL (Nagoya, Japan). Primary neuronal cultures were prepared from the cortices of C57BL/6 mice embryos at embryonic day 17 (E17) as described previously [25]. Cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite, Akita, Japan), and resuspended in nerve culture medium (Sumitomo Bakelite). Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6 mice at 14 days in vitro (DIV) using the “shaking off” method, which has been described previously [26]. Measurement of Soluble FKN Levels—Secreted soluble FKN from mouse primary microglia and cortical neurons was measured using an ELISA kit (R&D Systems) according to the TABLE 1 PCR primers and expected sizes of PCR products

Expected size

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS