The Research Of Autophagy In Hippocampal Neurons Of SMS2 Knockout Mice

Autophagy is a universal life phenomenon that exist in eukaryotic cells , of the last ten years, this is a focus in life sciences that is important process for cellular maintenance, cell viability, differentiation and development to degrade proteins and eliminate unwanted, damaged or redundant cell organelles or structures. More and more researchers noticed that autophagy is a key role in degrading misfolded and aggregation protein, which is closely related with neurodegenerative diseases . This topic is for the study of SMS2 knockout (SMS2^-/-)mice, by MAPLC3 and Beclin-1 immunocytochemistry, RT-PCR, electron microscopy, and Western blotting methods to investigate the impact of ceramide increased to autophagy, which is an important second messenger regulating cell apoptosis and to provide the basis for clinical treatment of neurodegenerative diseases by the induction of autophagy.To study the impact of ceramide increased to autophagy in SMS2^-/- mice hippocampal neurons, we bring to SMS2^+/- mice (two female, one male) from State University of New York of America and identified SMS2^-/- mice by PCR. then used SMS2^-/- mice as research object to explore the impact of ceramide to autophagy, so that to provide a theoretical basis of clinical treatment and prevention for neurodegenerative diseases.Objective : To investigate the impact of ceramide to autophagy in SMS2^-/- mice hippocampal neurons, In order to provide a theoretical basis of the prevention and clinical treatment for neurodegenerative diseases.Methods: SMS2^+/- mice were breed by the method of hybridism, backcross and cross, and mice genomic DNA were got out by phenol chloroform extraction method, the target gene were gained by PCR amplification, genotype of mice were identified by agarose gel electrophoresis, male and female homozygous SMS2^-/- mice were obtained, Then follow the same method of reproduction; To SMS2^-/- mice as a model group, the wild-type (WT) mice as the control group , autophagy and autophagosome-like structure can be seen in hippocampal neurons at P14 and P30 in control group and modle group by transmission electron microscopy; the number of MAPLC3-positive cells (PAC) in hippocampal neurons at P7, P14 and P30 in model group and control group were observed by immunofluorescence staining, and the co-expression levels in Beclin-1 and MAPLC3 in the nerve cells also can be seen; the protein level of MAPLC3 in hippocampal CA1 nerve cells were detected at P14 and P30 in model group and control group by Western blotting ; to analyze data by one-way ANOVA and LSD test.Results: 1. autophagy phenomenon in neurons in hippocampal of SMS2^-/- mice:â‘ With electron microscopy, a numerous lysosome and auto-phagosomes were found in the hippocampal neurons in SMS2^-/- mice .â‘¡Under light microscope, The positive autophagy cells (PAC) in modle were obviously higher than that in control at P7,P14 and P30 (P<0.01). 2. Accommodation of Beclin-1 to autophagy:â‘ the expression of Beclin-1 between in model group and control group are accordant: The Beclin-1 positive cells (BC) in model were obviously higher than that in control at P7,P14 and P30 (P<0.01) .â‘¡The co-expression of Beclin-1 and MAPLC3 in hippocampal neurons suggested the Beclin-1's regulation to the autophagy.â‘¢Western blotting analysis supported the results from immunocytochemistrys above.Conclusions: The number of ceramide were increased in neurons of SMS2^-/- mice , not only promotes apoptosis, at the same time promotes autophagy, to result in more autophagy in neurons .