Cold Exposure Interferes With Hippocampal Synaptic Plasticity And Energy Metabolism Through Ubiquitination

Cold stress is a common stressor,which extensively affects the functions of the immune system,neuroendocrine system,and other organs throughout the body.When faced with a cold environment,the body produces heat to maintain constant core body temperature.And the brain,as the commander of allocating energy supply to the whole body,will its own energy metabolism state be affected by cold stress? The hippocampus is an important central target of various stressors.Previous studies in this laboratory have found that chronic cold exposure can cause hippocampal neuron loss and cause behavioral changes in mice.Ubiquitination is a key mechanism that mediates neurodevelopment and synaptic plasticity.The mechanisms behind how protein ubiquitination affects neurodevelopment following cold exposure in the hippocampus of mice are not clear.Therefore,this study aims to explore the effects of cold exposure on the hippocampus of mice from the perspective of protein ubiquitination and pay attention to the changes in the energy metabolism of the hippocampus of mice under cold stress conditions.Five-week-old male C57BL/6 mice were randomly divided into a cold exposure group(CE)and a room temperature group(RT).The mice in the cold exposure group were exposed to a 4?artificial intelligence climate room for 3 hours a day,for 21 days.Nissl staining,Western blot,and immunohistochemical results showed that the number of surviving neurons in the hippocampal CA1 and CA3 areas of the cold exposure group was significantly reduced(P<0.05),and the expression level of microtubule-associated protein-2(MAP-2)protein was significantly down-regulated(P<0.05),the level of apoptosis-related protein cleaved-caspase-3 and the ratio of Bax/Bcl-2 were significantly up-regulated(P<0.05).These results indicate that chronic cold exposure negatively affects the neurodevelopment of the hippocampus of mice.To explore the effect of chronic cold exposure on protein ubiquitination in the hippocampus of mice,western blot was first used to detect the ubiquitin level of hippocampal tissue lysate.The results showed that chronic cold exposure significantly up-regulated the overall ubiquitin level of the hippocampus,as well as K48 polyubiquitin level,but down-regulated K63 polyubiquitin level.This suggests that ubiquitination is involved in the response of the hippocampus to cold exposure.Next,the label-free quantitative ubiquitination proteomics method was used to comprehensively screen the proteins whose ubiquitination level changes significantly under cold exposure conditions.The results showed that cold exposure induced significant changes in the ubiquitination levels of a series of synapse-related proteins in the hippocampus.Among them,the ubiquitination levels of postsynaptic density protein-95(PSD-95)at K202,K558,and K591 residues decreased significantly.Using the immunoprecipitation method,we found that cold exposure significantly down-regulated the ubiquitination level,K48 ubiquitination,and K63 ubiquitination levels of PSD-95.The results of Golgi staining showed that the dendritic spine density of neurons in the hippocampus CA1 area(P<0.001)and CA3 area(P<0.05)of the cold exposure group decreased significantly.It suggests that chronic cold exposure may inhibit the synaptic stabilization ability of PSD-95 by reducing the K63 ubiquitination level of PSD-95 at K558 residue,thereby reducing the density of hippocampal dendritic spines.The results of ubiquitination proteomics revealed that the ubiquitination levels of a variety of glucose metabolizing enzymes have undergone significant changes.To further explore the effect of cold exposure on the energy metabolism process of the hippocampus of mice,the hippocampus tissue was tested by targeted energy metabolomics.The results showed that the levels of malate and succinate in the tricarboxylic acid cycle pathway were significantly reduced,combined with the observation that gene expression levels of fumarate hydratase-1(FH1),succinate coenzyme A-ligase GDP forming subunit-?(SUCLG1),and succinate-coenzyme A-ligase GDP forming subunit-?(SUCLG2)were significantly decreased(P<0.05)detected by q-PCR method,indicating that chronic cold exposure inhibited the production of malate and succinate in the tricarboxylic acid cycle pathway.At the same time,in terms of mitochondrial function,cold exposure significantly down-regulated the activity of mitochondrial respiratory complex I(P<0.001),but had no significant effect on the activity of mitochondrial respiratory complex IV(P>0.05).Metabolomics results showed that the hippocampal FMN content decreased significantly.The q-PCR method detected a significant decrease in the m RNA level of NDUV1 subunit of mitochondrial respiratory complex I with FMN as a cofactor(P<0.001),and the m RNA level of NDUFV2 was also significantly down-regulated(P<0.001),while the gene expression level of NDUFS1 did not change significantly(P>0.05).In terms of glycolytic pathways,ubiquitination proteomics KEGG analysis indicated that differentially ubiquitinated proteins were significantly enriched in the important regulatory pathways of cell metabolism,HIF-1 signaling pathway and AMPK signaling pathway.Further tests showed that chronic cold exposure significantly upregulated phosphorylation level of m TOR(P<0.05)and expression level of HIF-1?(P<0.05),but down-regulated phosphorylation level of AMPK(P<0.001),indicating that chronic cold exposure activates the m TOR/HIF-1? pathway in the hippocampus.Ubiquitinomics data revealed that the ubiquitination level of pyruvate kinase M(PKM)at K3 residue was up-regulated,and the ubiquitination level at K135 residue was down-regulated.Further detection of PKM2 expression and pyruvate kinase activity,the results showed that PKM2 expression level and pyruvate kinase activity in the chronic cold exposure group were significantly decreased(P<0.05).At the same time,it was detected that the enzyme activity of lactate dehydrogenase(LDH)was up-regulated(P<0.01),and the enzyme activity of pyruvate dehydrogenase(PDH)was down-regulated(P<0.01).The above results suggest that chronic cold exposure interferes with hippocampal energy homeostasis.Conclusion: Chronic cold exposure can reduce hippocampal dendritic spine density by changing the level of PSD-95 ubiquitination.At the same time,chronic cold exposure interferes with the energy homeostasis of the hippocampus and induces changes in the ubiquitination of various glycolytic enzymes in the hippocampus.