The Role Of Anaphase-promoting Complex And Its Subunit Cdh1 In Ischemic Neuron Injury In Vivo And In Vitro.

Background and objectiveAnaphase-promoting complex (APC) is the major intracellular ubiquitin ligase that controls the cell cycle. Cdh1 is one of its co-activators, which is required for APC activity during late mitosis and G1. APC is activated by Cdh1 at late mitosis and the G1 phase, APC-Cdh1 is connected to the substrate proteins and transfed to ubiquitin, enableing ubiquitination of the substrate degradation by a protein enzyme body.This procession plays a key role in transformation from mitosis to G1 phase. Recent studies showed that APC-Cdh1 also expressed in mature neurons, it could regulate axon growth, synaptic transmission and neuronal survival. Our primary study showed, that APC-Cdh1 played an important role in the process in which neural stem cells differentiate into neurons; Reducing Cdh1 expression after spinal cord injury can promote axonal growth and neural function recovery. However, the role of APC-Cdh1 in the central nervous system injury and disease and its specific mechanism is not clear yet. Methods and Results1. Changes in the Expression of APC-Cdhl in Rats with focal cerebral ischemiaMethods:36 adult male Sprague-Dawley rats were randomly divided into two groups:①sham control group (n=12);②focal cerebral ischemia model group (n= 24), on which middle cerebral artery occlusion (MCAO) was produced by insertion of a nylon thread with rounded tip at bifurcation of right common carotid artery into internal carotid artery. At different times after injury, the expression of APC-Cdhl mRNA of brain tissue was detected by Real-time fluorescence quantitative PCR, double-label immunofluorescence assays was used to detecte the expression of Cdhl in different cells.Result:The expressions of Cdhl mRNA at each time point after surgery were lower than the sham group (P<0.05). Double immunofluorescence analysis showed that, Cdhl expressed abundantly in both injured and non-injured neurons, while Cdhl positive astrocytes were maimly observede in injured brain tissue.2. Expression of Cdhl and its downstream substrate during hypoxic injury of Primary cultured neuronsMethod:Primary neurons from cortex of postnatal 24h rat pups were cultured. Neuron was identified by NeuN immunocytochemical staining. The mRNA of neuron was extracted by Trizol method. The expression of Cdhl was examined by quantitive real time PCR. Using sugar-free Earle's solution and the three gas incubator Filled with nitrogen to maintain O2 concentration 1%, CO2 concentration of 5%,37℃for 1h. After reoxygenation treatment, Real-time quantitative PCR was used to detect the mRNA expression of Cdhl and its downstream substrates Skp2, Cyclin B1.Results:Rat primary neurons were uccessfully cultured, NeuN immunofluorescence showed about 90% of the cultured cells were neurons. OGD model of neuronal cells was established in vitro. At 3 days,7 days after the hypoxic injury in vitro, the expression of Cdhl and its downstream substrate Cyclin B1 in primary neurons were increased (P<0.05), while Skp2 expressions were lower at different time points after injury(P<0.05).3. Effects of lentivirus-mediated Cdhl RNAi in primary neuron survivalMethod:Primary neurons from cortex of postnatal 24h rat pups were cultured in the 24-hole cell culture plate.7days later, they were infected with lentivirus containing GFP in different MOI. Seventy-two hours later, the appropriate MOI for neuuons was determined by examining GFP expression with fluorescent microscope. Real-time quantitative PCR was used to detecte the expression of Cdhl. In this study, two groups were involved, uninfected group, and LV-Cdhl RNAi group. Cell apoptosis was evaluated TUNEL at 7dth after viral infection.Results:The appropriate lentivirus MOI for Primary cultured neurons was 100. The expression of Cdhl was significant reduced in LV-Cdhl RNAi group compaired to the uninfected group (P<0.05). TUNEL assay showed there was no significant difference in the absorption between the LV-Cdhl RNAi group and uninfected group (P>0.05)4. Statistical AnalysisAll of the statistical analyses were performed by SPSS 12.0 software. Measurement data were presented as means±SD. The differences between two groups were analyzed by t-test. The differences among three groups were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between different groups were analyzed by Chi-square test. P<0.05 was considered statistically significant. Conclusions1. APC-Cdhl in the normal rat brain tissue expressis mainly in neurons.After cerebral ischemic injury, the expression of neuronal Cdhl remains. Normal rat brain astrocytes are rare Cdhl-positive, while after ischemic injury, a large number of astrocytes were activated and were Cdhl-positive.2. In vitro experiments showed that OGD injury increased expression of neuronal Cdhl and its downstream substrate Cyclin B1, while Skp2 expression was reduced.3. Cdhl expression of Cdhl RNAi neuron group were reduced in vitro, but the lentivirus-mediated Cdhl RNAi didn't induce significant neuronal apoptosis.Research SummaryThe issue first revealed the expression of Cdhl in different nerve cells by in vivo experiments with hypoxic ischemic injury, that in normal brain tissue, Cdhl expressed mainly in neurons, but rare in astrocytes. Cdhl mRNA expression in ischemic tissue was decreased. A large number of astrocytes were activated and parts of them were Cdhl-positive. Then rat cortical neurons were cultured, and neuronal hypoxia model of neuronal hypoxia in vitro was built the in vitro. The Cdhl and Cyclin B1 mRNA expressions were found increased after injury, while the expression of Skp2 was decreased, suggesting that upregulation of Cyclin B1 might have induce the expression of Cdhl increasing by negative feedback. By lentivirus-mediated RNAi technology reduced the expression of neuronal Cdhl, no significant effect on cell survival in vitro was observed in short term. This study extended the understanding of Cdhl function in ischemic neural injury. It will be very useful for exploring the additional function of APC-Cdhl in nervous system injury and regeneration and gene therapies for neurological diseases.