Prenatal Alcohol Exposure Induces The Autophagy In Cerebellar Purkinje Cells Of Mice

Autophagy is a focus over the past decade in life sciences, which was another form of programmed cell death, to differentiate it from apoptoic cell death, named autophagic type-â…¡cell death. In mammals, autophagy is an important process for cellular maintenance, cell viability, differentiation and development to degrade proteins and eliminate unwanted, damaged or redundant cell organelles or structures. To explain the mechanisms of autophagy, some hypotheses were put forward, but the factors, such as the toxic effects of alcohol are still suspended, not to mention the understanding of its mechanism. Maternal alcohol exposure during gestation can cause serious injury to CNS of fetus. In this study, we observed the autophagy in cerebellar Purkinje cells at different ages (P0, P7, P14 and P30) mice by the model of prenatal alcohol-exposed mice, with electron microscopy, immunocytochemistry and Western blotting. Our results showed:1. Prenatal alcohol exposure and the development of Purkinje cells.In this study, we observed the difference between the control and models in development of Purkinje cells at P7, P14 and P30, With immunofluorescent labeling. This result showed that the difference between the ethanol-fed group and control group, such as the cortex more thinner, the cell smaller, cell alignment in a mess, the branches of dendrite decreased, the density of dendritic spine growed down, et al. And the change is more obviously in high-ethanol fed group. This shows that the development of Purkinje cells in pups were affect after prenatal alcohol, and there is does-dependent effect.2. With transmission electron microscopy confirmed the autophagy in Purkinje cells after prenatal alcohol exposure. The gold standard of evidence for autophagy should always be provided by ultrastructural support. Hence, the ultrastructures of Purkinje cells before and after prenatal alcohol exposure were analyzed to under transmission electron microscope. Comparing with control and models, we found the many autophagolysosomes could appear in the Purkinje cells after prenatal alcohol exposure, especially in High-does alcohol group. The autophagical Purkinje cells usually companied other ultrastuctural alterations, such as swelled mitochondria with cristae damage, extensive capsule in Golgi complexes. The lysosomes also increased greatly in the autophagical Purkinje cells. This result conformed that the adverse effects in development of Purkinje cells in pups by ultrastructures, and it could be induced the occurrence of be autophagy after prenatal alcohol.3. Purkinje cells'autophagy under light microscope and Western blotting.The microtubule-associated light chain 3 (LC3) is an essential component of autophagy that acts as an adaptor protein between microtubules and autophagosomes through its N-terminal domain. It has been shown to be associated with autophagic membranes and thus can be used as autophagosomal markers. In this study, we observed the autophagy in Purkinje cells under light microscope with immunofluorescent labeling, including single labeling of MAPLC3, double labeling of MAPLC3 and Calbindin. This result showed that the positive autophagy cells (PAC) and positive autophagy index (PAI) in ethanol treatment groups were obviously higher than that in control at either P7 or P14 with dose dependency (P<0.001). In the meantime, PAC and PAI increased gradually with age increasing (F=58.29, P<0.001). But, PAC and PAI were no obvious difference between alcohol-treatment group and control group at P30 (P>0.05). Western blottings analysis supported the results from immunocytochemistrys above. This shows that the prenatal ethanol exposure can induce the autophagy of Purkinje cells with does-dependency and long-term effects. 4. Beclin-1 and regulation to autophagy.Beclin-1 was a specific gene in mammals which regulated autophagy. It was conformed that beclin-1 is an important positive regulator factor of autophagy. With single labeling of Beclin-1, double labeling Calbindin and double labeling MAPLC3, we found Beclin-1 was expression in ethanol model group and control group were as same MAPLC3, and they are co-expressed in the same cells nearly, and there is a little of expression of MAPLC3 in the nucleus, but Beclin 1 did not expressed. That is to say the expression both MAPLC3 and Beclin-1 in the same cells were positive correlation. Western blottings analysis supported the results from immunocytochemistrys above. From this result, we can conclude the effect which alcohol induces the autophagy in Purkinje cells is regulated by Beclin-1 and when the autophagy-related genes, such as MAPLC3, are activated by ethanol or its metabolic product aldehyde, the autophagy would be upregulated by Beclin-1.As we all know that autophagy is a physiologic adjustment mode of life derived from the nature, it is an essential instrument in the process of cell renewing and resisting to the bad environment. Through this study, we would explain the relevant mechanisms, which for people are more deeply understanding the harm of maternal alcohol abuse to generations, especially to the development of fetuses or children cerebellum. It is also helpful for us to understand the pathogenesis of alcohol-related neurological diseases such as Wernicke encephalopathy or alcoholic cerebellar degeneration. So, there were a huge social benefit of improving people's physique and promotion of prenatal care, and it had some theoretical value for the developmental neurobiology, neurology and neurological toxicology.