title: The Toxic Effect of ALLN on Primary Rat Retinal Neurons.
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.
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Shang
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China.
authors.forename: Shu-Chao
authors.initials: SC
authors.lastname: Wang
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China.
authors.forename: Lv-Shuang
authors.initials: LS
authors.lastname: Liao
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China. huangjufang@csu.edu.cn.
authors.forename: Ju-Fang
authors.initials: JF
authors.lastname: Huang
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, 410013, China. xiongkun2001@163.com.
authors.forename: Kun
authors.initials: K
authors.lastname: Xiong
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D005455:Fluorescent Antibody Technique; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007976:Leupeptins / Q000633:toxicity*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D055351:Retinal Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D053148:Caspase 3; D007770:L-Lactate Dehydrogenase; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C494828:Bax protein, rat; C000617196:Bcl2 protein, rat; C526109:Anxa3 protein, rat; C063261:acetylleucyl-leucyl-norleucinal; C505410:Casp3 protein, rat
keywords: ALLN; Apoptosis; Necrosis; Retinal neurons; Toxicity