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Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells

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Abstract

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

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Acknowledgments

This work was supported by Department of Biotechnology, Government of India, grant no.:BT/PR14241/MED/30/423/2010. We would like to thank Satish Sagar for his assistance with the figures.

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Authors and Affiliations

  1. Neurobiology Biology Laboratory, School of Biotechnology, KIIT University, Bhubaneswar, 751024, India

    Premranjan Kumar, Mitali Madhusmita Swain & Arttatrana Pal

  2. School of Chemical and Biotechnology, SASTRA University, Thanjavur, India

    Thiagarajan Raman

  3. Division of Oncology, National Jewish Health, Denver, CO, USA

    Rangnath Mishra

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ESM 1

Supplementary Table 1. Sequences of the primer pairs used in qRT-PCR (DOC 58 kb)

ESM 2

Supplementary Fig. 1. Hyperglycemia induces DNA damage in PC-12 cells. a Comet assay showing the DNA damage in PC-12 cells cultured in a medium containing without or with HG (25 mM) for 24 h. b Agarose gel electrophoresis analysis shows an increased DNA fragmentation of PC-12 cells cultured in a medium containing in HG for 24 h (PPT 278 kb)

ESM 3

Supplementary Fig. 2. Hyperglycemia decreases the levels of antioxidant enzymes in PC-12 cells. a Antioxidants (catalase [CAT], superoxide dismutase [SOD], and glutathione [GSH]) were significantly decreased in diabetic brain compared with control rats. Spectrophotometric assay shows a significant decrease in the activities of antioxidants (CAT, SOD, and GSH) in PC-12 cells in a dose- (b) and (c) time-dependent manner of glucose. d A representative immunoblot analysis of CAT and SOD in PC-12 cells exposed to different concentrations of glucose. e Exogenous NAC restores the antioxidants activity (*p < 0.05 and **p < 0.01) (PPT 308 kb)

ESM 4

Supplementary Fig. 3. Hyperglycemia induces lipid peroxidation (LPO) and protein carbonylation (PC) in neuronal cells. a Exposure of PC-12 cells to different concentrations of additional glucose significantly increases the level of LPO and PC in concentration-dependent manner for 24 h and b HG (25 mM) glucose for 3–48 h. c Pretreatment of NAC to PC-12 cells decreases the HG-induced LPO and PC production (*p < 0.05 and **p < 0.01) (PPT 211 kb)

ESM 5

Supplementary Fig. 4. Hyperglycemia induces iNOS expression in diabetic brain. Immunohistochemistry analysis shows a significant increase in iNOS expression in diabetic brain compared with control rats (PPT 808 kb)

ESM 6

Supplementary Fig. 5. Hyperglycemia induces chromosomal aberration in PC-12 cells. a Karyotyping images depicted a representative mitotic metaphase chromosomes spread in HG-treated cells. b G-bending assay in PC-12 cells treated with HG (PPT 335 kb)

ESM 7

Supplementary Fig. 6. Hyperglycemia induces MMP-2/9 expression in PC-12 cells. PC-12 cells were treated with different concentration glucose for 24 h, and then, the samples were prepared for zymogram. The zymography analysis was carried out for MMP-2/9 molecules (PPT 77 kb)

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Kumar, P., Raman, T., Swain, M.M. et al. Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells. Mol Neurobiol 54, 238–254 (2017). https://doi.org/10.1007/s12035-015-9667-3

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