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The Toxic Effect of ALLN on Primary Rat Retinal Neurons

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Abstract

N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 μM, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 μM but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 μM. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 μM after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 μM, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 81371011, 81571939); National Key Technologies Research and Development Program of China (2012BAK14B03); Wu Jie-Ping Medical Foundation of the Minister of Health of China (320.6750.14118); Natural Science Foundation of Hunan Province (2015JJ2187); Teacher Research Foundation of Central South University (2014JSJJ026); and the Project of Innovation-driven Plan of Central South University (2015CXS022).

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  1. Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China

    Na Li, Lei Shang, Shu-Chao Wang, Lv-Shuang Liao, Dan Chen, Ju-Fang Huang & Kun Xiong

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  1. Na Li
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  2. Lei Shang
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Correspondence to Ju-Fang Huang or Kun Xiong.

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Li, N., Shang, L., Wang, SC. et al. The Toxic Effect of ALLN on Primary Rat Retinal Neurons. Neurotox Res 30, 392–406 (2016). https://doi.org/10.1007/s12640-016-9624-6

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  • DOI: https://doi.org/10.1007/s12640-016-9624-6

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