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Oxidative Stress and Autophagic Alteration in Brainstem of SOD1-G93A Mouse Model of ALS

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Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease involving both upper and lower motor neurons. The mechanism of motor neuron degeneration is still unknown. Although many studies have been performed on spinal motor neurons, few have been reported on brainstem and its motor nuclei. The aim of this study was to investigate oxidative stress and autophagic changes in the brainstem and representative motor nuclei of superoxide dismutase 1 (SOD1)-G93A mouse model of ALS. The expression levels of cluster of differentiation molecule 11b (CD11b), glial fibrillary acidic protein, glutamate–cysteine ligase catalytic subunit, heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, voltage-dependent anion-selective channel protein 1, Sequestosome 1/p62 (p62), microtubule-associated protein 1 light chain 3B (LC3), and SOD1 proteins in brainstem were examined by Western blot analysis. Immunohistochemistry and immunofluorescence were performed to identify the cellular localization of SOD1, p62, and LC3B, respectively. The results showed that there were progressive asctrocytic proliferation and microglial activation, induction of antioxidant proteins, and increased p62 and LC3II expression in brainstem of SOD1-G93A mice. Additionally, SOD1 and p62 accumulated in hypoglossal, facial, and red nuclei, but not in oculomotor nucleus. Furthermore, electron microscope showed increased autophagic vacuoles in affected brainstem motor nuclei. Our results indicate that brainstem share similar gliosis, oxidative stress, and autophagic changes as the spinal cord in SOD1-G93A mice. Thus, SOD1 accumulation in astrocytes and neurons, oxidative stress, and altered autophagy are involved in motor neuron degeneration in the brainstem, similar to the motor neurons in spinal cord. Therefore, therapeutic trials in the SOD1G93A mice need to target the brainstem in addition to the spinal cord.

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Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (no. 81171210) and Science and Technological Department of Hebei Province (no. 11966122D).

Conflict of interest

All authors read, approved the manuscript, and declared no competing financial interests.

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Author notes

  1. Ting An

    Present address: Department of Neurology, Beijing Shunyi Hospital of China Medical University, Beijing, 101300, China

Authors and Affiliations

  1. Department of Neurology, The Second Hospital of Hebei Medical University, 215 Heping West Road, Shijiazhuang, Hebei Province, 050000, China

    Ting An, Pengxiao Shi, Weisong Duan, Shipan Zhang, Pin Yuan, Zhongyao Li, Dongxia Wu, Chunyan Li & Yansu Guo

  2. Key Laboratory of Hebei Neurology, Shijiazhuang, Hebei, 050000, China

    Weisong Duan, Zhongyao Li, Dongxia Wu, Chunyan Li & Yansu Guo

  3. Institute of Cardiocerebrovascular Disease, Shijiazhuang, Hebei, 050000, China

    Weisong Duan, Chunyan Li & Yansu Guo

  4. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605, USA

    Zuoshang Xu

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Fig. S1

Histological localization of hypoglossal nucleus (12 N), facial nucleus (7 N), oculomotor nucleus (3 N) and red nucleus (RMC). (A) NeuN immunohistochemistry showing hypoglossal nucleus; (B) NeuN immunohistochemistry showing facial nucleus; (C) SMI-32 immunohistochemistry showing oculomotor nucleus and red nucleus. Right column are Images extracted from the mouse brain in stereotaxic coordinates of Paxinos and Franklin [17] (JPEG 7 kb)

High-resolution image (TIFF 2,894 kb)

Fig. S2

Progressive gliosis, oxidative stress and autophagic changes with the disease progression in different parts of brainstem in SOD1-G93A mice. (A) Immunoblotting analysis of CD11b, GFAP and HO-1 expression. (B) Immunoblotting analysis of p62, LC3, VDAC1 and hSOD1 expression. All data are representative of three independent experiments. Mb, Midbrain; Po, Pons; Mo, medulla oblongata. 1, non-transgenic littermate; 2, presymptomatic stage; 3, onset stage; 4, end stage (JPEG 4 kb)

High-resolution image (TIFF 748 kb)

Fig. S3

Beclin-1 and Atg-5 expression in different parts of brainstem of non-transgenic littermates and SOD1-G93A mice at end stage. (A) Western blot analysis of beclin-1 and Atg-5 expression. (B) Quantitative analysis of (A). Mb, Midbrain; Po, Pons; Mo, medulla oblongata. No significant difference was observed, compared with non-transgenic littermates (JPEG 13 kb)

High-resolution image (TIFF 3767 kb)

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An, T., Shi, P., Duan, W. et al. Oxidative Stress and Autophagic Alteration in Brainstem of SOD1-G93A Mouse Model of ALS. Mol Neurobiol 49, 1435–1448 (2014). https://doi.org/10.1007/s12035-013-8623-3

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