Mechanism Of CK2 Mediated NR2B Phosphorylation By Inhaled Anesthetics In Neonatal Neurotoxicity

Mechanism of NR2B phosphorylated by Inhaled Anesthetics induce developing neuron impairmentResearch Background and PurposeDuring the development of nerve system, never cells are always in a imbalanced state between excitement and inhibition. During the nerve developmental phase, there lacks the effect of inhibitory neurotransmitter, nevertheless the excitatory neurotransmitter-GABA has an important role both in neuron growth and synaptic formation in the same period, which seems could promote the neurons growth and plasticity, whereas no pathological changes could be detected. The cause maybe lies in the special feature of NMDA receptor.In forebrain area, in where hippocampus exists, the proportion of NMDA receptor:NR2A/NR2B increases during the progression of embryonic development. In the meantime the LTP and LTD’ s induction effects change correspondingly. All these indicate that the proportion of NR2A/NR2B can impact brain development and synapses formation. NR2A and NR2B both are the subunit of NMDA receptor. In the process of neuron development, part NR2B transformed into NR2A. Researches indicate that the transformation is regulated by kinase PKA, PKC and CK2. NR2B can protect the neuron damage from euphoria. In the nerve development phase, postsynaptic membrane mainly express NR2B. In postnatal phase, with the maturity of the nerve system, receptor NR2B is phosphorylated, which induced endocytosis. Receptor NR2A is inserted in the postsynaptic membrane in the same time. Gradually NR2B is replaced by NR2A, the synapses of young nerve cells mature.P35, an regulating agonist of Cdk5, is a brain specificity of calcium protease substrate. Both P35 and Cdk5 are important channel proteins maintaining brain development and survival. By activating NMDAR of primary nerve cell, glutamate can stimulate calcium dependent calcium protease mediated hydrolysis, which turn p35 into smaller molecular p25. Cdk5 abnormal activation and target reset can induce neuronal apoptosis. Inhibition of calcium protease or Cdk5 can prevent neuron injury mediated by p25. Moreover, neurons which experienced some form of nonagitation-toxic injury also need p25 generated by calcium protease mediation. This indicate that the potential treatment effects of calcium protease inhibitors are not only limited to the toxic damage of neurons. In partial ischemic cerebral apoplexy and total brain ischemic mice model, as well as patients’ tissue samples of Alzheimer’s disease and ischemic stroke the expression of p25 is increase. This is consistent with its important role in toxicity of excitement. It is important that over expression of p25 in forebrain of transgenic mouse will trigger nerve degeneration, inhibit Cdk5 of its downstream genes, prevent damage of neurons caused by p25 and cerebral ischemia. Though there are many proteins could be substrate of Cdk5 phosphorylation, p25 induced neuron damage was proved to be cause by:Cdk5 mediated phosphorylation of GluN2A, the subunit of NMDAR; muscle cells enhancement factor 2; antioxidant enzyme Prx2, no purine/de-oxidation pyrimidine endonuclease phosphorylation; suppression and inhibition of histone deacetylase.Our lab’s recently study show that inhalation anesthetics facilitate voltage dependent calcium channels (VDCC) and N-methyl-D-aspartic acid (NMDA) in puberty neurons, increase the concentration of free calcium ion of neurons, lead to obvious toxic effects. Further study of inhaled general anesthetics caused neurons synaptic plasticity, its mechanism and prevention measures is especially important to guide the clinical safety use of inhaled anesthetics. This study intends to confirm the following hypothesis:1. Isoflurane affects developing neurons’form and function by changing the NMDAR phosphorylation level and the proportion of its subtypes.2. Isoflurane affect NMDAR receptor structure and function by CK2-CDK5-p35 pathway.3. There is no significant effect of clinical relevant concentration isoflurane on puberty long-term learning memory function of rats.Methods and Results1. Determinate the NMDA subunit expression change in hippocampal neurons after inhaling isoflurane. Use SD rats in the same litter 5 days postnatal, randomized into two groups. The control group executed immediately after inhaling air, while treatment group executed after inhaling same concentration of isoflurane (MAC 1.3) for2,4,6 hours respectively, separate hippocampus. Ser 1480 phosphorylation is an important regulation target of NR2B receptor, NR2B phosphorylation induced endocytosis and insertion of NR2A receptor. By using Western Blot method, to detect changes of NR2A and NR2B protein expression and Ser 1480 phosphorylation level in hippocampal neurons before and after the anesthetic isoflurane inhaling.Result:1.3% Isoflurane dose-dependent reduces NR2B-Ser1480 and NR2B membrane expression, P<0.05. NR2A and NR2B total protein show no significant difference between groups. And the NR2B-PSD95 complex decrease in Iso-4h group, P<0.05..2. Determinate isoflurane inhaling effects on CK2 signal pathway in development of hippocampal neurons.Choose SD rats in the same litter 5 days postnatal, randomized into two groups. The control group executed immediately after inhaling air, while treatment group executed after inhaling same concentration of isoflurane (MAC 1.3) for 2.4,6 hours respectively, separate hippocampus. By using Western Blot method, to detect CK2, p35/p25 protein expression level in hippocampal neurons. Result:1.3% Isoflurane dose-dependent reduces CK2 expression and increases p35/p25 expression, P<0.05.3. Determinate isoflurane inhaling effects on hippocampal neuron apoptosis and Caspase-3 expression.Choose SD rats in the same litter 5 days postnatal, randomized into three groups. Isoflurane group inhale same concentration of isoflurane (MAC 1.3) for 4 hours, Isoflurane+Minocycline group given Minocycline treatment before isoflurane inhalation, while control group inhale fresh air. Execute rats immediately after the treatment, taking out the intact brain tissue to make the hippocampus frozen section slides. Detecting the hippocampus neuron cell number with Caspase 3 activity by using immunofluorescence method. Result:Activated Caspase-3 cell in Iso-4h group are much more than those in controls, P<0.05, which can be inhibited by Minocycline.4. Determinate CDK5 inhibitor Roscovitine’s effect on rat’s hippocampal neurons expression of NMDAR after isoflurane inhalation. Choose SD rats in the same litter 5 days postnatal, randomized into four groups. Isoflurane group inhale same concentration of isoflurane (MAC 1.3) for 4 hours, Isoflurane+Roscovitine group given Roscovitine treatment before isoflurane inhalation, while Roscovitine group only given Roscovitine treatment and control group only inhale fresh air. Execute rats immediately, separate and extract proteins of hippocampus. By using Western Blot method, to detect NR2A, NR2B, p-NR2B (S1480) protein expression level in hippocampal neurons.Result:NR2B and NR2B-Ser1480 expression on membrane increase significantly in Roscovitine+Iso group than Iso group, P<0.05.5. Determinate CDK5 inhibitor Roscovitine’s effect on CDK5-p35-CK2 signal pathway in rat’s hippocampal neurons after isoflurane inhalation. Choose SD rats in the same litter 5 days postnatal, randomized into four groups. Isoflurane group inhale same concentration of isoflurane (MAC 1.3) for 4 hours, Isoflurane+Roscovitine group given Roscovitine treatment before isoflurane inhalation, while Roscovitine group only given Roscovitine treatment and control group only inhale fresh air. Execute rats immediately, separate and extract proteins of hippocampus. By using Western Blot method, to detect CK2, p35/p25 protein expression level in hippocampal neurons.Result:CK2 expression increases in Roscovitine+Iso group than Iso group, P<0.05.6. Determinate CDK5 inhibitor Roscovitine’s effect on p-NR2B (S1480) in different regions of rat’s hippocampus after isoflurane inhalation. Choose SD rats in the same litter 5 days postnatal, randomized into four groups. Isoflurane group inhale same concentration of isoflurane (MAC 1.3) for 4 hours, Isoflurane+Roscovitine group given Roscovitine treatment before isoflurane inhalation, while Roscovitine group only given Roscovitine treatment and control group only inhale fresh air. Execute rats immediately, taking out the intact brain tissue to make the hippocampus frozen section slides. Detecting the p-NR2B (S1480) expression in DG, CA1, CA3 region of hippocampus respectively by using immunohistochemical method.Result:NR2B-Ser1480 cell counting reduces significantly in Iso group than control group and Roscovitine+Iso group in CA1 region of hippocampus.7. The forward neural toxicity studies. Choose SD rats in the same litter 5 days postnatal, randomized into two groups.The treatment group inhale same concentration of isoflurane (MAC 1.3) for 2, 4,6 hours respectively under the blood oxygen saturation monitoring, while the control group inhale fresh air. Continue to feed both groups for 8 weeks in the same circumstance. Compare the learning and memory function difference between two groups by Barnes Maze and Fear Conditioning behavioral test. Then compare the difference of hippocampus neuron number, synapses number, and its form and related protein expression changes between two groups.Result:The latency to target box in Iso-6h group increases significantly than in the other groups in both tests, P<0.05.Statistical AnalysisAll data were analyzed by SPSS 13.0 statistical software, measurement data using mean±standard deviation (x±s), one-way ANOVA was used for comparison in group, paired t-test were used for comparison between groups, P<0.05 for the statistically significant difference.Conclusions1.Exposure to clinical relevant concentration isoflurane dose-dependent regulates the composition of NMDAR and ratio of subunits and phosphorylation of NR2B in neonatal rats, impact the developing neuron morphology and function.2. Prove that isoflurane modulates NMDAR formation and function through CDK5-p35-CK2 signalling pathway.3. Behavior tests of spatial learning and memory shows no significant effects when exposing to lower dose clinical relevant concentration isoflurane.Summary1. Isoflurane down-regulates CK2, which abnormally activate CDK5-p35, and induce the phosphorylation of NR2B at Tyr1472, lead to endocytosis of NR2B. Decreased CK2 also reduces phosphorylation of NR2B at Ser1480, disrupts the combination of NR2B and PSD95,reduces the NR2B expression on membrane.2. Behavior tests of spatial learning and memory shows that proper dose clinical relevant concentration isoflurane seems have no obvious impairment in long term neurotoxicity of development rats.