Rat C6 glioma cells were obtained from JCRB cell bank (Osaka, Japan) and were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. Mitotracker Red CMXRos Mitochondria Probe was purchased from Lonza Walkersville, Inc (Walkersville, MD, USA). Horseradish peroxidase-linked secondary antibodies were from Zymed Laboratories (South San Francisco, CA, USA). Anti-LC3B antibody and anti-GABARAPL2 (GATE-16) antibody was from MBL (Nagoya, Japan). Materials and methods MaterialsĬCCP and an anti-actin antibody were from Sigma (St. In addition, we examined the effects of CCCP, which causes mitochondrial damage, on the expression of GABARAPL2 in C6 glioma cells. In this study, rat C6 glioma cells, which are often used as a model for astrocytes, were used to observe the expression of the mRNA of all of the members of the LC3 subfamily and GABARAP subfamily. However, the functions of GABARAPL2 in damaged mitochondria remain unclear. Nix and GABARAPL1 are engaged in mitophagy by carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Nix, which is an outer mitochondrial membrane protein, binds LC3A, GABARAP, GABARAPL1, and GABARAPL2 but only weakly binds LC3B. The degradation of damaged mitochondria is considered as the selective autophagy (mitophagy). The degradation of cellular contents by autophagy occurs through both nonselective and selective pathways. Although the physiological functions of the members of the LC3 subfamily, except for LC3B, and the GABARAP subfamily are not clear, the members of the LC3 subfamily and GABARAP subfamily are linked to PE, and they play a role in the formation of autophagosomes. LC3B-PE (LC3B-II) plays a crucial role in the formation of the autophagosome, and the conversion of unconjugated-LC3B (LC3B-I) to LC3B-II is the index of the induction of autophagy. LC3B is catalyzed by ATG4 and is then conjugated to phosphatidylethanolamine (PE) by the ATG12–ATG5–ATG16 complex. Among the LC3 subfamily and the GABARAP subfamily, LC3B is most commonly used for studying autophagy. In rats, the LC3 subfamily includes LC3A and LC3B, and the GABARAP subfamily includes GABARAP, GABARAP-like 1 (GABARAPL1), and GABARAP-like 2 (GABARAPL2, which is also known as GATE-16). The autophagosome is formed during the stage of elongation and closure, and the important proteins that form autophagosomes are the members of the microtubule-associated protein 1 light chain 3 (LC3) subfamily and gamma-aminobutyric-acid-type-A receptor-associated protein (GABARAP) subfamily. In the process of autophagy, cellular cytosolic components are isolated by the autophagosome. The processes in autophagy include the stages of initiation, elongation and closure, and maturation. Macroautophagy (hereafter referred to as autophagy) is the bulk degradation process of cellular cytosolic components, such as defective proteins and damaged organelles. Therefore, the isolation and removal of damaged mitochondria are necessary to maintain the function of astrocytes. This mitochondrial damage leads to the dysfunction and death of astrocytes. Ischemia, hypoxia and traumatic injury cause oxidative stress which results in mitochondrial damage. All rights reserved.Astrocytes function to form the blood–brain barrier, provide nutrients to neurons, retain the extracellular ion balance, surround synapses, and process information with neurons. In particular, we analyze the cellular modifications produced by the activation of two major pathways involving the signaling of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the transcription factor EB (TFEB), and discuss the contribution of these pathways to the integrated cellular stress response.ĬCCP CCCP, Carbonyl Cyanide m Chlorophenyl Hydrazone (PubChem CID: 2603) FCCP, Carbonyl Cyanide p Trifluoromethoxyphenylhydrazone (PubChem CID: 3330) Mitochondria Mitophagy N-Acetyl-l-cysteine (PubChem CID: 12035) NRF2 TFEB.Ĭopyright © 2018 Elsevier Inc. We here review current mechanistic insights into the induction of mitophagy and autophagy by CCCP and FCCP. Sustained perturbation of ΔΨm, which is normally tightly controlled to ensure cell proliferation and survival, triggers various stress pathways as part of the cellular adaptive response, the main components of which are mitophagy and autophagy. CCCP and FCCP are now being increasingly used for investigating the mechanisms of autophagy by inducing mitochondrial degradation through the disruption of the mitochondrial membrane potential (ΔΨm). The ring-substituted derivatives of carbonyl cyanide phenylhydrazone, CCCP and FCCP, are routinely used for the analysis of the mitochondrial function in living cells, tissues, and isolated mitochondrial preparations.

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