GSK-3? Negatively Regulate The Necroptosis In The Basolateral Amygdala Of Aged Mice

Background:Normal aging is associated with a series of pathological changes.Neuronal loss is one of the most prominent pathological hallmarks during aging.Neuronal loss was found in the hippocampus,basolateral amygdala and ventral tegmental area of aging mice.Apoptosis and necroptosis are the two main cell death types that lead to neuron loss in the aged brain.Necroptosis is a programmed form of necrosis and is activated in the adipose tissue of the aged mice and increases with age.The activation of necroptosis promotes the loss of hippocampal neurons in AD model mice.However,whether the activation of necroptosis is involved in the loss of basolateral amygdala neurons in aged mice is still unclear.In addition,the activation of necroptosis can promote aging-related pathological changes such as inflammation,axonal degeneration,and cognitive and motor dysfunction.However,the underlying mechanisms that regulate the activation of the necroptosis during aging remain unclear.In this study,we use the senescence accelerated mice prone 8(SAMP8)mice,a well-established mouse model of age-related phenotypes,to explore the activation of necroptosis in the amygdala of aged mice and its mechanism.Objective:To explore the activation of necroptosis in the amygdala of aged mice and related mechanism.Methods:In this experiment,SAMP8 mice and the control group SAMR1 mice,GSK-3?^flox/+mice and C57/BL6 mice were used.The immunohistochemistry,immunofluorescence and immunoblotting experiments were used to detect that whether the occurrence of the necroptosis in the amygdala of those mice.Screened by m RNA microarrays and RT-PCR validation,GSK-3?was associated with the necroptosis in the amygdala of SAMP8 mice.Through stereotactic injection of adeno-associated virus and lentivirus into the BLA of the mice to further verify the relation of GSK-3?and the necroptosis.Results:The density of neurons decreases with age and the necroptosis increases with age in the amygdala of SAMP8 mice.The necroptosis in the BLA of SAMP8 mice is significantly activated from 5 months,which promotes neuron loss;By using m RNA microarray,we identified GSK-3?was highly correlated with the necroptosis in the BLA of aged mice.Moreover,genetic knockdown or artificially inhibition of GSK-3?led to the activation of the neuronal necroptosis and restoring the GSK-3?in the BLA of SAMP8 mice inhibited the activation of the neuronal necroptosis in aged mice;GSK-3?regulated RIPK3-dependent necroptosis by regulating m TORC1 signaling pathway.Gene deletion of GSK-3?in the BLA of wild-type mice activated the m TORC1 signaling pathway,which in turn led to autophagy dysfunction and RIPK3accumulation.Overexpression of GSK-3?in the BLA of SAMP8 mice inhibited the m TORC1 signaling pathway and reduced the level of RIPK3.We further identified a long noncoding RNA,termed GSK-3?-AS,to regulate negatively GSK-3?m RNA and protein expression;In addition,social isolation accelerated activation of the necroptosis in 4-month-old SAMP8 mice,accompanied by the abnormal GSK-3?-AS/GSK-3?/m TORC1 signals.Conclusion:1)The necroptosis is significantly activated in the BLA of SAMP8 mice from 5 months,which promotes neuron loss;2)The decrement of GSK-3?in the BLA of the aged mice was positively correlated with the necroptosis.Moreover,genetic deletion or silencing of GSK-3?led to neuronal necroptosis in the BLA,while overexpression of GSK-3?inhibited neuronal necroptosis in the BLA of aged mice;3)The loss of GSK-3?led to the activation of m TORC1 signal and then induced the RIPK3-dependent necroptosis in the BLA.Non-coding RNA GSK-3?-AS mediated transcriptional repression of GSK-3?in aged mice;4)Social isolation accelerated the neuronal necroptosis in the BLA of 4-month-old SAMP8 mice,accompanied by abnormal GSK-3?-AS/GSK-3?/m TORC1 signals.