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The induction of neuronal death by up-regulated microglial cathepsin H in LPS-induced neuroinflammation.

Fan K, Li D, Zhang Y, Han C, Liang J, Hou C, Xiao H, Ikenaka K, Ma J - J Neuroinflammation (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. fankaijunjun@hotmail.com.

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.

No MeSH data available.


Related in: MedlinePlus

Microglial Cat H expression was induced at a transcriptional level after LPS injection. ISH staining showed that at 6 h after LPS injection, Cat H mRNA expression was detected in the hippocampus (D), SNr (E), and cerebral cortex (F). At 72 h after LPS injection, the intensity and density of Cat H mRNA positive cells were both increased in the above regions (G-I). In the control, brain Cat H mRNA was absent in the brain (A-C), present only close to the blood vessels (arrows in J). Double staining (Cat H ISH following Iba-1 IHC) showed that cellular localization of Cat H mRNA was perivascular microglia (arrows in L). At 24 h after LPS injection, Cat H mRNA positive signals appeared in microglia in the brain parenchyma, in addition to perivascular microglia (arrows in K, M). The quantification results of Cat H mRNA expression in brain regions analyzed after LPS injection were shown in (N). The detected regions were illustrated in sagittal section of the brain (O). Data were expressed as mean + SEM from three independent experiments. aP, fP, gP < 0.05 vs control; bP < 0.01 vs 6 h; cP < 0.05 vs 72 h; dP < 0.05 vs 24 h; eP < 0.01 vs 6 h. Scale bar = 50 μm (A, B, D, E, G, H); 20 μm (C, F, I); 10 μm (J; K, L, M). n = 5.
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Fig1: Microglial Cat H expression was induced at a transcriptional level after LPS injection. ISH staining showed that at 6 h after LPS injection, Cat H mRNA expression was detected in the hippocampus (D), SNr (E), and cerebral cortex (F). At 72 h after LPS injection, the intensity and density of Cat H mRNA positive cells were both increased in the above regions (G-I). In the control, brain Cat H mRNA was absent in the brain (A-C), present only close to the blood vessels (arrows in J). Double staining (Cat H ISH following Iba-1 IHC) showed that cellular localization of Cat H mRNA was perivascular microglia (arrows in L). At 24 h after LPS injection, Cat H mRNA positive signals appeared in microglia in the brain parenchyma, in addition to perivascular microglia (arrows in K, M). The quantification results of Cat H mRNA expression in brain regions analyzed after LPS injection were shown in (N). The detected regions were illustrated in sagittal section of the brain (O). Data were expressed as mean + SEM from three independent experiments. aP, fP, gP < 0.05 vs control; bP < 0.01 vs 6 h; cP < 0.05 vs 72 h; dP < 0.05 vs 24 h; eP < 0.01 vs 6 h. Scale bar = 50 μm (A, B, D, E, G, H); 20 μm (C, F, I); 10 μm (J; K, L, M). n = 5.

Mentions: In this study, we first investigated the Cat H expression at transcriptional level in the normal brain. The ISH results showed that Cat H mRNA was absent in the brain parenchyma, only present in cells with glial morphology, close to the blood vessels (Figure 1A-C,J), also in non-parenchymal sites, such as leptomeninges and the choroid plexus in ventricles.Figure 1


The induction of neuronal death by up-regulated microglial cathepsin H in LPS-induced neuroinflammation.

Fan K, Li D, Zhang Y, Han C, Liang J, Hou C, Xiao H, Ikenaka K, Ma J - J Neuroinflammation (2015)

Microglial Cat H expression was induced at a transcriptional level after LPS injection. ISH staining showed that at 6 h after LPS injection, Cat H mRNA expression was detected in the hippocampus (D), SNr (E), and cerebral cortex (F). At 72 h after LPS injection, the intensity and density of Cat H mRNA positive cells were both increased in the above regions (G-I). In the control, brain Cat H mRNA was absent in the brain (A-C), present only close to the blood vessels (arrows in J). Double staining (Cat H ISH following Iba-1 IHC) showed that cellular localization of Cat H mRNA was perivascular microglia (arrows in L). At 24 h after LPS injection, Cat H mRNA positive signals appeared in microglia in the brain parenchyma, in addition to perivascular microglia (arrows in K, M). The quantification results of Cat H mRNA expression in brain regions analyzed after LPS injection were shown in (N). The detected regions were illustrated in sagittal section of the brain (O). Data were expressed as mean + SEM from three independent experiments. aP, fP, gP < 0.05 vs control; bP < 0.01 vs 6 h; cP < 0.05 vs 72 h; dP < 0.05 vs 24 h; eP < 0.01 vs 6 h. Scale bar = 50 μm (A, B, D, E, G, H); 20 μm (C, F, I); 10 μm (J; K, L, M). n = 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Microglial Cat H expression was induced at a transcriptional level after LPS injection. ISH staining showed that at 6 h after LPS injection, Cat H mRNA expression was detected in the hippocampus (D), SNr (E), and cerebral cortex (F). At 72 h after LPS injection, the intensity and density of Cat H mRNA positive cells were both increased in the above regions (G-I). In the control, brain Cat H mRNA was absent in the brain (A-C), present only close to the blood vessels (arrows in J). Double staining (Cat H ISH following Iba-1 IHC) showed that cellular localization of Cat H mRNA was perivascular microglia (arrows in L). At 24 h after LPS injection, Cat H mRNA positive signals appeared in microglia in the brain parenchyma, in addition to perivascular microglia (arrows in K, M). The quantification results of Cat H mRNA expression in brain regions analyzed after LPS injection were shown in (N). The detected regions were illustrated in sagittal section of the brain (O). Data were expressed as mean + SEM from three independent experiments. aP, fP, gP < 0.05 vs control; bP < 0.01 vs 6 h; cP < 0.05 vs 72 h; dP < 0.05 vs 24 h; eP < 0.01 vs 6 h. Scale bar = 50 μm (A, B, D, E, G, H); 20 μm (C, F, I); 10 μm (J; K, L, M). n = 5.
Mentions: In this study, we first investigated the Cat H expression at transcriptional level in the normal brain. The ISH results showed that Cat H mRNA was absent in the brain parenchyma, only present in cells with glial morphology, close to the blood vessels (Figure 1A-C,J), also in non-parenchymal sites, such as leptomeninges and the choroid plexus in ventricles.Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. fankaijunjun@hotmail.com.

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.

No MeSH data available.


Related in: MedlinePlus