Ruifang Qi1, Xiaolu Zhang2, Yabin Xie2, Shuyuan Jiang2, You Liu2, Xiaolei Liu2, Wei Xie2, Xiaoe Jia2, Rengui Bade2, Ruili Shi2, Sijie Li3, Changhong Ren3, Kerui Gong4, Chunyang Zhang5, Guo Shao6. 1. Department of Neurobiology and Center of Stroke, Beijing Institute for Brain Disorders, School of Basic Medical Science, Capital Medical University, Beijing, China; Inner Mongolia Key laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, China; Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China. 2. Inner Mongolia Key laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, China; Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China. 3. Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China. 4. Department of Oral and Maxillofacial Surgery, University of California San Francisco, San Francisco, USA. 5. Department of neurosurgery, the First Affiliated Hospital of Baotou Medical College, Inner Mongolia, China. 6. Department of Neurobiology and Center of Stroke, Beijing Institute for Brain Disorders, School of Basic Medical Science, Capital Medical University, Beijing, China; Inner Mongolia Key laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, China; Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China. Electronic address: shao.guo.china@gmail.com.
Abstract
BACKGROUND: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. METHODS: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. RESULTS: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. CONCLUSIONS: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.
BACKGROUND: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. METHODS: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. RESULTS: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. CONCLUSIONS: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.
Authors: Anastasia V Graf; Maria V Maslova; Artem V Artiukhov; Alexander L Ksenofontov; Vasily A Aleshin; Victoria I Bunik Journal: Int J Mol Sci Date: 2022-02-25 Impact factor: 5.923
Authors: Cristina Gallego-Fabrega; Elena Muiño; Jara Cárcel-Márquez; Laia Llucià-Carol; Miquel Lledós; Jesús M Martín-Campos; Natalia Cullell; Israel Fernández-Cadenas Journal: Int J Mol Sci Date: 2022-06-20 Impact factor: 6.208