NR2B Mediated Effects Of Dysfunction Of Ubiquitin-proteasome System Components On CREB Activity

The majory function of ubiquitin-proteasome system (UPS) is to regulate variety of biological processes through degradating intracellular aggregation or misfolded proteins, and depend on this process the normal physiological state and functions of cells can be maintained. The components of UPS dysfunction have a seriously negative impact on the organism, especially when the occurrence of these abnormal effects in central nervous system would induce the lost expression of memory-related proteins or even impaire the maintenance of synaptic plasticity and memory function which further cause memory decline or neurodegenerative disorders. N-Methyl-D-aspartate (NMDA) receptor NR2B subunit plays extremely biological functions in synaptic signaling events, protein-protein interactions and human neurodegeneration. A growing body of evidence suggests that many substrates of UPS have an effect on NR2B biological function, including microtubule-associated protein tau, synaptic proteins, protein kinases and phosphatase and so on, thus a consideration is suggested that UPS probably regulates NR2B subunit through its substrates and further affects synaptic plasticity and learning and memory.The current research focused on cAMP response element binding protein (CREB), and the molecular mechanisms of dysfunctional UPS components on the regulation of memory function were explored from the levels of cells, hippocampal slices to animals. As a transcription factor, CREB is crucial for regulating the expression of neuronal proteins which is required for the synaptic plasticity and formation and maintenance of memory. In addition to the proteasome, the transcriptional activity of CREB can also be modulated by the downstream molecular signaling of NMDA receptor. Therefore, to explore the molecular mechanisms of dysfunctional UPS components on the regulation of CREB activity are an effective pathway to learning and memory function.1. Proteasome inhibition induces CREB dephosphorylation (Cell lines, N2a cells). The current study indicated that proteasome activity inhibition down-regulated the level of phosphorylated CREB at Ser133site via two pathways which further decreased CREB activity.(1) Inhibition of proteasome activity caused abnormal phosphorylation and ubiquitination of tau protein. Moreover, the abnormal tau protein affected the normal physiological function of NMDA receptor NR2B subunit by down-regulating the level of phosphorylated NR2B at Tyr1472site which eventually led to a significant decrease in phosphorylation of CREB at Ser133site.(2) Inhibition of proteasome activity induced the alternation of protein kinase A (PKA) and glycogen synthase kinase-3β (GSK-3(3) activities which further resulted in an increase in dephosphorylation of CREB-Ser133.2. Inhibition of deubiquitination enzyme ubiquitin C-terminal hydrolase L1(UCH-L1) is involved in CREB dephosphorylation in hippocampal slices (organotypic hippocampal slices, slices from C57BL/6mice). Researches have demonstrated that UCH-L1have an effect on phosphorylation and transcriptional activity of CREB, but the mechnisams still unclear. Therefore, this current research used UCH-L1inhibitor LDN to treat mice hippocampal slices and explored the effects of UCH-L1inhibition on CREB activity. The results indicated that inhibition of UCH-L1activity induced a significant reduction of phosphorylated CREB-Ser133. Meanwhile, the hyperphosphorylation of tau protein, down-regulation of tyrosin kinase Fyn activity, reduction of phosphorylated NR2B at Tyr1472site and increased level of synaptic protein PSD-95(postsynaptic density protein95) were observed after UCH-L1inhibition. Hypephosphorylated tau protein probably disturbed the formation of Fyn/NR2B/PSD-95complex to affect the biological activity of NR2B subunit and ultimately led to decreased CREB activity. Moreover, inhibition of UCH-L1activity induced reductive PKA activity also involved in the regulation of CREB activity.3. The molecular mechanisms of dysfunctional proteasome and UCH-L1affect the spatial memory in mice (Animal, APP/PS1double transgenic mice). APP/PS1double transgenic mouse is an Alzheimer’s disease (AD) mouse model and the mainly pathological characteristic of the mouse is the deposition of senile plaque which has cytotoxicity. The results showed that APP/PS1mice have significant spatial memory deficits. Further detection found that compared with the control group, CREB activity in hippocampus and cortex in APP/PS1mice significantly reduced. In addition, the alterations of the molecular pathways that regulated the CREB activity were observed in APP/PS1mice. Firsly, the obviously decreased level of brain-derived neurotrophic factor (BDNF) was closely related to the down-regulation of CREB activity; secondly, hyperphosphorylated tau protein had an effect on the phosphorylation of NR2B at Tyr1472site which was likely to decrease the normal physiological function of NR2B subunit and further resulting dephosphorylation of CREB. Additonally, proteasome and UCH-L1were involved in the regulation of the activity of above-mentioned molecular pathways. However, in the current study, both the expressions and activities of proteasome and UCH-L1were significantly decreased in APP/PS1mice, indicating the defective spatial learning and memory in APP/PS1might be caused by down-regulation of CREB activity which induced by dysfunctional ubiquitin-proteasome system components. Therefore, according to the above results (part1to3), our study from proteasome and UCH-L1to abnormal tau protein and to NR2B/CREB this molecular pathway revealed the mechanisms of impaired spatial memory function of APP/PS1mouse.