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. 2008 Nov 12;582(27):3823-31.
doi: 10.1016/j.febslet.2008.10.015. Epub 2008 Oct 21.

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Arjun Saha  1 , Sung-Jo Kim, Zhongjian Zhang, Yi-Ching Lee, Chinmoy Sarkar, Pei-Chih Tsai, Anil B Mukherjee
Affiliations

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Arjun Saha et al. FEBS Lett. .

Abstract

Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca(2+)-homeostasis and stimulates the expression of Ca(2+)-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca(2+)-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL.

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Figures

Fig. 1
Fig. 1
Elevated expression of S100B and RAGE in the brain tissues of PPT1-KO mice and their WT littermates. Expression of S100B- (A) and RAGE- (D) mRNAs in the brain of 1, 3 and 6-month old PPT1-KO mice and in those of their WT littermates. Western blot analyses of brain lysates from PPT1-KO mice and their WT littermates showing elevated expression of S100B- (B) and RAGE (E) in the PPT1-KO mouse brain. Western blot analyses of S100B (C) and RAGE (F) in postmortem brain tissue lysates from a normal control and an INCL patient. The bar graphs below the Western blots represent densitometric quantitation of the protein bands. The error bars indicate the standard deviation of the mean (n=3). Asterisks indicate statistical significance: *, p< 0.010; **, p< 0.005. AU, arbitrary units.
Fig. 2
Fig. 2
Expression of S100B and RAGE in cultured astroglial cells from PPT1-KO mice and WT littermates. Expression of S100B- (A) and RAGE- (C) mRNAs in WT and PPT1-KO astroglial cells. Western blot analyses of S100B (B) and RAGE (D) in WT and PPT1-KO astroglial cells. Quantitation (bar graphs) is provided under each Western blot. Error bars indicate standard deviation of the mean (n=3). Asterisks indicate statistical significance at p<0.005.
Fig. 3
Fig. 3
The activation of Src-kinase, MEK1/2, ERK1/2 and p38 MAP kinases in astroglial cells from the PPT1 KO mice. (A) Western blot analyses of WT and PPT1-KO astroglial cell extracts using anti-phospho-Src antibody; anti-c-Src was used as the control. (B) Western blot analyses of WT and PPT1-KO astroglial cell extracts using anti-phospho-MEK1/2 antibody; anti-MEK1/2 was used as the control. (C) Western blot analyses of WT and PPT1-KO astroglial cell extracts using anti-phospho-ERK1/2 antibody; anti- ERK1/2 was used as the control. (D) Western blot analyses of WT and PPT1-KO astroglial cell extracts using anti-phospho-p38 antibody; anti- p38 was used as the control. Note the elevation of Src- and MAPK- phosphorylation in PPT1-KO astroglial cells. The error bars indicate standard deviation of the mean (n=3).
Fig. 4
Fig. 4
Increased phosphorylation of IkBα and nuclear translocation of NF-kB p65 subunit in PPT1-KO astroglial cells. (A). Western blot analyses of astroglial cell extracts from PPT1-KO and WT littermates using phospho-IkBα antibody showing increased phosphorylation of IkBα in PPT1-KO astroglial cells. (B) Western blot analyses of nuclear extracts from PPT1-KO and WT astroglial cells using NF-kB p65 antibody. Histone H4 was used as the loading standard for the nuclear protein fractions (C) Activation of NF-kB in cultured astroglial cells from the PPT1-KO and WT mice. The cells were processed for immunofluorescence staining detecting sub-cellular localization of NF-kB subunit p65 (red) in the WT (Left panels) and PPT1-KO (Right panels). Nuclei were stained with DAPI (blue; middle panel). Increased immunoreactivity for p65 in the nuclei of KO cells was also observed. Scale bars, 100μm. The error bars indicate standard deviation of the mean (n=3).
Fig. 5
Fig. 5
Elevated secretion of IL-1ß, IL-6, MCP-1 and TNFα in PPT1-KO mouse brain extract as well as in those of the PPT1-KO conditioned media from astroglial cell cultures. The results are expressed as the mean (n=3) ±SD.
Fig. 6
Fig. 6
Effect of a RAGE-specific siRNA transfection of PPT1-deficient astroglial cells to suppress RAGE expression. Both WT and PPT1-KO astroglial cells were transfected with siRNAs or with a scrambled siRNA (Ambion, USA) as control. Western blot analyses were performed using anti-RAGE , -ß-Actin, -pSrc, c-Src, -pERK1/2, -ERK1/2, -pP38 or –P38 antibodies.
Fig. 7
Fig. 7
Effect of RAGE-SiRNA on the expression of pro-inflammatory cytokines. Note that suppression of RAGE-expression by SiRNA markedly decreased the cytokine production.
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