title: The induction of neuronal death by up-regulated microglial cathepsin H in LPS-induced neuroinflammation.
abstract: BACKGROUND
  Neuroinflammation is a hallmark that leads to selective neuronal loss and/or dysfunction in neurodegenerative disorders. Microglia-derived lysosomal cathepsins are increasingly recognized as important inflammatory mediators to trigger signaling pathways that aggravate neuroinflammation. However, cathepsin H (Cat H), a cysteine protease, has been far less studied in neuroinflammation, compared to cathepsins B, D, L, and S. The expression patterns and functional roles of Cat H in the brain in neuroinflammation remain unknown.
  
  
  METHODS
  C57BL/6J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze expression and localization of Cat H in the brain. Nitrite assay was used to examine microglial activation in vitro; ELISA was used to determine the release of Cat H and proinflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ). Cat H activity was analyzed by cellular Cat H assay kit. Flow cytometry and in situ cell death detection were used to investigate neuronal death. Data were evaluated for statistical significance with one-way ANOVA and t test.
  
  
  RESULTS
  Cat H mRNA was only present in perivascular microglia and non-parenchymal sites under normal conditions. After LPS injection, Cat H mRNA expression in activated microglia in different brain regions was increased. Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level. The expression of Cat H protein exhibited the similar alterations after LPS injection. In vitro, inflammatory stimulation (LPS, TNF-α, IL-1β, IL-6, and IFN-γ) increased the release and activity of Cat H in microglia. Conversely, addition of Cat H to microglia promoted the production and release of NO, IL-1β, and IFN-γ which could be prevented by neutralizing antibody. Further, addition of Cat H to Neuro2a cells induced neuronal death.
  
  
  CONCLUSIONS
  Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation. The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.
authors.affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. fankaijunjun@hotmail.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Fan
authors.affiliation: Clinical Medicine of Seven-year Education, Dalian Medical University, Dalian, 116044, , Liaoning, China. lidaobo19930211@hotmail.com.
authors.forename: Daobo
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. zyl2080@126.com.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Regenerative Medicine Center, the First Affiliated Hospital, Dalian Medical University, Dalian, 116011, , Liaoning, China. hanchao0427@163.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Han
authors.affiliation: Graduate School, Dalian Medical University, Dalian, 116044, , Liaoning, China. lcwapple@qq.com.
authors.forename: Junjie
authors.initials: J
authors.lastname: Liang
authors.affiliation: Graduate School, Dalian Medical University, Dalian, 116044, , Liaoning, China. dlhcy1990@163.com.
authors.forename: Changyi
authors.initials: C
authors.lastname: Hou
authors.affiliation: Graduate School, Dalian Medical University, Dalian, 116044, , Liaoning, China. xylsy2006@163.com.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki, 444-8787, , Aichi, Japan. ikenaka@nips.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ikenaka
authors.affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. ma_jianmei@hotmail.com.
authors.forename: Jianmei
authors.initials: J
authors.lastname: Ma
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000906:Antibodies / Q000627:therapeutic use; D001921:Brain / Q000378:metabolism*; D056655:Cathepsin H / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004660:Encephalitis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D008070:Lipopolysaccharides / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009573:Nitrites; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D000906:Antibodies; D016207:Cytokines; D008070:Lipopolysaccharides; D009573:Nitrites; D056655:Cathepsin H; D010751:Phosphopyruvate Hydratase