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HMGB1 Promotes Mitochondrial Dysfunction-Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation.

Qi L, Sun X, Li FE, Zhu BS, Braun FK, Liu ZQ, Tang JL, Wu C, Xu F, Wang HH, Velasquez LA, Zhao K, Lei FR, Zhang JG, Shen YT, Zou JX, Meng HM, An GL, Yang L, Zhang XD - PLoS ONE (2015)

Bottom Line: Glycyrrhizin also significantly inhibited 3-NP-induced striatal damage.It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1.These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center, Cyrus Tang Medical Institute, Soochow University, Suzhou, Jiangsu, China.

ABSTRACT
Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP-induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP-induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

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HMGB1 knockdown in primary striatal neurons without 3-NP treatment.(A,B) Primary striatal neurons were transduced with lentiviral vectors expressing either non-targeted control shRNA (Con-shRNA) or one of two different shRNAs targeting HMGB1 expression (HMGB1-shRNA1 and HMGB1-shRNA2) and cultured for 48 h. Total cellular extracts were subjected to Western blotting for HMGB1, LC3, SQSTM1, p-JNK, and JNK expression analysis. Densities of protein bands were analyzed with an image analyzer (Sigma Scan Pro 5) and normalized to the loading control (β-actin). Bars represent mean ± SE; n = 3 samples per group. Groups were compared by ANOVA followed by Dunnet’s post hoc test before data conversion. **p <0.01 vs control shRNA.
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pone.0142901.g004: HMGB1 knockdown in primary striatal neurons without 3-NP treatment.(A,B) Primary striatal neurons were transduced with lentiviral vectors expressing either non-targeted control shRNA (Con-shRNA) or one of two different shRNAs targeting HMGB1 expression (HMGB1-shRNA1 and HMGB1-shRNA2) and cultured for 48 h. Total cellular extracts were subjected to Western blotting for HMGB1, LC3, SQSTM1, p-JNK, and JNK expression analysis. Densities of protein bands were analyzed with an image analyzer (Sigma Scan Pro 5) and normalized to the loading control (β-actin). Bars represent mean ± SE; n = 3 samples per group. Groups were compared by ANOVA followed by Dunnet’s post hoc test before data conversion. **p <0.01 vs control shRNA.

Mentions: To analyze the apparent impact of HMBG1 on autophagy and apoptosis signaling, we used a lentiviral shRNA approach to target HMGB1 expression in neuronal cells, including primary striatal neurons. Striatal neurons were infected by one of two HMGB1-targeting shRNAs or control shRNA followed by a selection for stable clones. Clones transduced with either of the HMGB1-targeted shRNAs had reduced expression of HMGB1 whether treated with 3-NP or not (Figs 4A and 5A). The decrease in LC3-II and increase in p62 after HMGB1 knockdown indicated that HMGB1 is involved in the regulation of basal autophagy without treatment of 3-NP. Similar to the incubation with the HMGB1 inhibitor glycyrrhizin, shRNA-mediated knockdown of HMGB1 expression significantly reduced 3-NP–mediated effects on autophagy and apoptosis as shown by reduced JNK phosphorylation, LC3-II activation, and decline of SQSTM1 (Fig 5A and 5B) with 3-NP treatment.


HMGB1 Promotes Mitochondrial Dysfunction-Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation.

Qi L, Sun X, Li FE, Zhu BS, Braun FK, Liu ZQ, Tang JL, Wu C, Xu F, Wang HH, Velasquez LA, Zhao K, Lei FR, Zhang JG, Shen YT, Zou JX, Meng HM, An GL, Yang L, Zhang XD - PLoS ONE (2015)

HMGB1 knockdown in primary striatal neurons without 3-NP treatment.(A,B) Primary striatal neurons were transduced with lentiviral vectors expressing either non-targeted control shRNA (Con-shRNA) or one of two different shRNAs targeting HMGB1 expression (HMGB1-shRNA1 and HMGB1-shRNA2) and cultured for 48 h. Total cellular extracts were subjected to Western blotting for HMGB1, LC3, SQSTM1, p-JNK, and JNK expression analysis. Densities of protein bands were analyzed with an image analyzer (Sigma Scan Pro 5) and normalized to the loading control (β-actin). Bars represent mean ± SE; n = 3 samples per group. Groups were compared by ANOVA followed by Dunnet’s post hoc test before data conversion. **p <0.01 vs control shRNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4643922&req=5

pone.0142901.g004: HMGB1 knockdown in primary striatal neurons without 3-NP treatment.(A,B) Primary striatal neurons were transduced with lentiviral vectors expressing either non-targeted control shRNA (Con-shRNA) or one of two different shRNAs targeting HMGB1 expression (HMGB1-shRNA1 and HMGB1-shRNA2) and cultured for 48 h. Total cellular extracts were subjected to Western blotting for HMGB1, LC3, SQSTM1, p-JNK, and JNK expression analysis. Densities of protein bands were analyzed with an image analyzer (Sigma Scan Pro 5) and normalized to the loading control (β-actin). Bars represent mean ± SE; n = 3 samples per group. Groups were compared by ANOVA followed by Dunnet’s post hoc test before data conversion. **p <0.01 vs control shRNA.
Mentions: To analyze the apparent impact of HMBG1 on autophagy and apoptosis signaling, we used a lentiviral shRNA approach to target HMGB1 expression in neuronal cells, including primary striatal neurons. Striatal neurons were infected by one of two HMGB1-targeting shRNAs or control shRNA followed by a selection for stable clones. Clones transduced with either of the HMGB1-targeted shRNAs had reduced expression of HMGB1 whether treated with 3-NP or not (Figs 4A and 5A). The decrease in LC3-II and increase in p62 after HMGB1 knockdown indicated that HMGB1 is involved in the regulation of basal autophagy without treatment of 3-NP. Similar to the incubation with the HMGB1 inhibitor glycyrrhizin, shRNA-mediated knockdown of HMGB1 expression significantly reduced 3-NP–mediated effects on autophagy and apoptosis as shown by reduced JNK phosphorylation, LC3-II activation, and decline of SQSTM1 (Fig 5A and 5B) with 3-NP treatment.

Bottom Line: Glycyrrhizin also significantly inhibited 3-NP-induced striatal damage.It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1.These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center, Cyrus Tang Medical Institute, Soochow University, Suzhou, Jiangsu, China.

ABSTRACT
Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP-induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP-induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

Show MeSH
Related in: MedlinePlus