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Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 ( Ndufv1 ) and Ndufv2 . In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Stress is a nonspecific response to adverse circumstances and chronic stress can destroy homeostasis, leading to various primary diseases. Although chronic cold stress is becoming increasingly important for individuals living or working in extreme environments, the risk of associated disorders of the central nervous system remains unstudied. Here, male C57BL/6 mice were exposed to a temperature of 4 °C, for three hours each day for one, two or three weeks. Glial cell activation, neuronal structure, and neuroinflammation were then evaluated by western blotting, immunofluorescence, Nissl staining and co-immunoprecipitation. Microglial activation, accompanied by activation of the NF-κB signaling pathway, release of pro-inflammatory cytokines and loss of Nissl bodies, was observed in mouse hippocampal tissue following cold exposure. We speculate that these phenomena are mediated by the HMGB1/TLR4/NF-κB pathway and closely associated with acetylation of HMGB1 in the hippocampus. These findings provide new insights into the mechanisms of the cold stress response, which should inform the development of new strategies to combat the effects of hypothermia., (Copyright © 2019. Published by Elsevier B.V.)

Cold stress can induce neuroinflammation in the hippocampal dentate gyrus (DG), but the mechanism underlying neuronal apoptosis induced by cold stress is not well-understood. To address this issue, male and female C57BL/6 mice were exposed to a temperature of 4 °C for 3 h per day for 1 week, and glial cell activation, neuronal apoptosis, and neuroinflammation were evaluated by western blotting, immunofluorescence, terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling, Nissl staining, and immunohistochemistry. Additionally, BV2 cells were treated with different concentrations of cortisol (CORT) for 3 h to mimic stress and molecular changes were assessed by western blotting, immunofluorescence, and co-immunoprecipitation. We found that excess CORT activated glial cells and increased neuroinflammation in the DG of mice exposed to cold temperatures, which was associated with increased acetylation and nuclear factor-κB signaling. These effects were mediated by the acetylation of lysine 9 of histone 3 and lysine 310 of p65, which resulted in increased mitogen-activated protein kinase phosphorylation, nuclear translocation of p65, microglia activation, and acetylation of high-mobility group box 1. Neuroinflammation was more severe in male compared to female mice. These findings provide new insight into the mechanisms of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.

Stress induces many non-specific responses in the hippocampus, especially during adolescence. Low environmental temperature is known to induce stress, but its influence on the hippocampus, especially in adolescent mice is not clear. We compared apoptotic-related protein levels and MAPK signaling pathway activation in hippocampal neurons of adolescent mice under low temperature conditions (4 °C for 12 h) with western blotting and immunohistochemistry. Western bolt results demonstrated that the levels of phospho-JNK, phospho-p38, and cleaved-caspase 3 significantly increased, while the ratio of Bcl-XL/Bax decreased, in the cold stress group. The results of immunohistochemistry (IHC) and Nissl staining demonstrated that the protein optical density of caspase 3 increased and Nissl bodies decreased in the cold stress group compared with controls. Thus, we conclude that cold exposure initiates activation of the MAPK signaling pathway and subsequently induces the upregulation of pro-apoptotic proteins in the hippocampi of adolescent mice. Overall our study reveals the relationship between cold stress and apoptosis in adolescent mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Stress induces many different sex-specific physiological and psychological responses during adolescence. Although the impact of certain brain stressors has been reported in the literature, the influence of cold stress on the mechanisms underlying hippocampal neurotransmitter disorder and neuroinflammation remain unstudied. Adolescent male and female C57BL/6 mice were exposed to 4 °C temperatures, 3 h per day for 1 week. Serum CORT and blood gas analysis was then used to assess body status. Using western blotting, immunofluorescence and immunohistochemistry we also assessed glial cell number and microglial activation, as well as inflammatory cytokine levels and related protein expression levels. The phenomena of excessive CORT, microglial activation, increased acetylate-HMGB1 levels, NF-κB signaling pathway activation, pro-inflammatory cytokine release, neuronal apoptosis and neurotransmitter disorder were demonstrated in mouse hippocampal tissue following cold exposure. We believe that these phenomena are mediated by the HMGB1/TLR4/NFκB pathway. Finally, the male inflammatory response in hippocampal tissue was more severe and the influence of cold exposure on neurotransmitter was greater in females., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Stress induces many non-specific inflammatory responses in the mouse brain, especially during adolescence. Although the impact of stress on the brain has long been reported, the effects of cold stress on hippocampal neuroinflammation in adolescent mice are not well understood; furthermore, whether these effects are gender specific are also not well established. Adolescent male and female C57BL/6 mice were exposed to 4 °C temperatures for 12 h, after which behavior was assessed using the open field test. Using western blotting and immunohistochemistry we also assessed glial cell numbers and microglial activation, as well as inflammatory cytokine levels and related protein expression levels. We found that in mice subjected to cold stress: 1) There were significant behavioral changes; 2) neuronal nuclei densities were smaller and total cell numbers were significantly decreased; 3) nuclear factor (NF)-κB and phosphorylated AKT were upregulated; 4) pro-inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α were also upregulated; and 5) microglia were activated, while glial fibrillary acid protein and ionized calcium-binding adapter molecule 1 protein expression increased. Taken together, these results indicate that cold stress induces pro-inflammatory cytokine upregulation that leads to neuroinflammation and neuronal apoptosis in the hippocampi of adolescent mice. We believe that these effects are influenced by a GABAB/Rap1B/AKT/NF-κB pathway. Finally, male mice were more sensitive to the effects of cold stress than were female mice., (Copyright © 2018 Elsevier Inc. All rights reserved.)