The Role Of Autophagy In The Cold-stress Response In Zebrafish Cells

Autophagy(or autophagocytosis)refers to the process that some cytoplasmic protein or origanelle packaged by double-membrane vesicle,fuses with lysosomes and the contents are degraded and recycled.Inessential proteins and dysfunctional components are degraded for remaining cell's survival when exposed to extreame environment like nutrient starvation.Whereas in some cases excessive autophagy may induce apoptosis and promote cell death.Did the autophagy participate in cold resistant or antifreeze of Antarctic fish is unknown,despites the molecular mechanisms and function of autophagy are studied clearly.Thus,it is a new object and is important for clarify the molecular mechanisms about the process of Antarctic fish's cold resistant or antifreeze.In this study,a putative autophagosome maker LC3(microtubule-associated protein 1 light chain 3)was used to monitor autophagy in cells under cold stress.pEGFP-LC3 ? m Cherry-h LC3B-pcDNA3.1 were transfected into ZF4 cells and significant autophagy was observed,EGFP-LC3 puncta were observed in ZF4 cells after cold treatment,indicating that ZF4 cells underwent autophagy under cold stress in a time-dependent manner and reached its peak 48 hr after treatment.To exclude the possibility that accumulation of EGFP-LC3 puncta was a result of slowed autophagic flux due to low temperature,lysotracker red was used to show increased autolysosome under cold shock.At the same time,wild-type ZF4 cells were exposed to cold pressure,and endogenous LC3 activation was investigated.Under cold pressure,increased expression and cleavage of LC3 was observed.Meanwhile,expression of autophagy-related members was examined using real-time quantitative reverse transcription PCR,the mRNA levels of LC3 and beclin 1 increased under cold stress.Above data indicated that ZF4 cells underwent autophagy under cold stress.Cell viability assay showed that ZF4 cells showed decreased cell growth and cell viability under cold stress.To investigate the role of autophagy in ZF4 cells under cold stress,an autophagy inhibitor bafilomycin A1,which inhibits autophagosome�lysosome fusion,and a shRNA plasmid targeted against beclin-1 were used.Beclin-1 shRNA could efficiently downregulate the expression of beclin-1,and compared to scrambled shRNA,beclin-1 shRNA could decrease cell viability remarkably under cold shock.At the same time,bafilomycin A1 co-treatment also decreased cell viability under cold shock.Overexpression of beclin 1 could protect cells form cold-induced cell death.Above data suggest autophagy play a protective role in ZF4 cells under cold shock.It has been reported that autophagy can protect cells from ROS induced cell death via eliminating dysfunctional mitochondria,the major source of ROS.So the effect of autophagy on ROS production and mitochondria was detected.First,we found acute cold shock could induce significant ROS production,and NAC could decreased cell death,indicating that cold shock induced ROS,and then ROS contributed to cell death.It is interesting that NAC treatment also decreased the accumulation of autophagosomes and autolysosome,indicating that ROS is require for induction of autophagy under cold shock.Then we found that bafilomycin A1 treatment induced the level of ROS was significant increased,indicating that autophagy was participate in ROS clear for high level ROS may cause DNA damage and cell death.The relationship between autophagy and ROS was investigated in this study.First we found NAC treatment attenuates cold-induced autophagy,indicated that ROS is a critical activator of autophagy in ZF4 cells under sub-lethal cold stress.Then we found that bafilomycin A1 treatment increased ROS level in ZF4 cells under cold stress,indicating that autophagy in turn could reduce ROS level.It has been reported that autophagy can prevent ROS overproduction by eliminate dysfunctional mitochondria,a major source of ROS production.Co-localization of mitochondrial and LC3 could be observed in ZF4 cells under cold stress,indicating the elimination of dysfunctional mitochondria under cold stress.