The Study On The Effect Of ApoE4on Rat Hippocampal Long-term Synaptic Plasticity And Its Molecular Mechanism

Alzheimer disease (AD) is a chronic, primary and irreversibleneurodegenerative disorder characterized by deterioration of cognitive and mentalfunction, including learning and memory[1]. Four genes have been reported to beassociated with AD phenotypes, including the amyloid precursor protein (APP)[2,3],presenilin-1(PS1)[4], presenilin-2(PS2)[5,6] and apolipoprotein-E4(APOE4)genes[7,8]. APOE4is a major risk factor for late-onset family AD. It is reported thatthe increased risk for AD is approximately3.6times and8times in people with oneε4allele and two ε4alleles, respectively, compared to individuals with no ε4alleles[7,9].The apolipoprotein E4(apoE4) which translated from the APOE4gene hasstrong neurotoxicity. The influence of apoE4protein on AD has been clearlydemonstrated. For example, apoE4increased Aβ aggregation[10,11], facilitated tauhyperphosphorylation[12,13], suppressed the activities of delayed-rectifier potassium(IK) channels[14], and decreased spine density[15], dendritic complexity and brainmetabolism[16-18]. ApoE4also induced inflammatory responses in cultured cells andtargeted replacement mice expressing human E4(apoE4-TR)[19-23]. In addition,over expression of apoE4impaired spatial memory and avoidance memory in mice[24-27].However, the effects of apoE4on synaptic plasticity are highly controversial inthe in vitro experiments with brain slices of apoE4-overexpressing or apoE-deficientmice [28-30]. It is also an open question whether and how the apoE4affects the invivo hippocampal late-phase long-term potentiation (L-LTP), a well-establishedelectrophysiological cellular model for learning and memory. In addition, theassociation of apoE4with Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB), two critical protein kinase ortranscription factor in the LTP induction and maintenance, is mostly unknown. So,the present study observed the neurotoxicity of apoE4on the long term synapticplasticity and investigated its probable mechanisms. The study includes two parts:(1)by using an in vivo electrophysiological field potential recording technique, we testedthe effects of acute application of apoE4on the induction and maintenance of in vivoL-LTP in the hippocampal CA1region of rats.(2) After L-LTP experiments, theconcentrations of CaMKII and CREB in the hippocampus of rats were examined byusing immunochemistry and western blotting techniques so as to clarify the molecularmechanisms underlying the action of apoE4on L-LTP.PartⅠ ApoE4Impaired Hippocampal Late-Phase Long TermPotentiation in Rat Hippocampal CA1Region in vivoTo characterize the neurotoxicity role of apoE4in the brain, we investigated theeffects of intrahippocampal injection of apoE4on synaptic placiticity of the rats byin vivo electrophysiological recording of L-LTP in hippocampal CA1region. Adultmale SD rats were used. Under anesthesia with urethane, hippocampal fEPSP wererecorded in the CA1region. In addition, the L-LTP was induced by three groups ofhigh frequency stimulation (HFSs). ApoE4solution was injected into thehippocampus at different time. The paired pulse facilitation (PPF) in the CA1regionof rats was also recorded.The results showed that:(1) the drugs in different group in the experimentdidn’t change the base line synaptic transmission.(2) three groups of HFSssuccessfully induced hippocampal L-LTP in control group and apoE2group. AveragefEPSP amplitudes were211.0±7.2%,170.4±4.5%and161.2±3.3%immediately,60min and180min post-HFSs in the control group and210.8±8.4%,168.4±4.9%and157.75±3.6%in the ApoE4group at the same time points (P>0.05). In the pre-HFSsinjection group,0.2μg apoE4inhibited the induction and maintenance of L-LTP, thefEPSPs being178.3±5.8%,149.7±5.0%and142.2±5.0%at the same time points ascontrol group(P<0.01)。(3) In post-HFSs injection groups, the same concentration (0.2μg) of apoE4produced a slight, but not significant decrease in the average fEPSPamplitude, being155.5±6.5%and143.5±7.9%at60min and180min post-HFSs,respectively (p>0.05)。Further, injection of high dose (2μg) ApoE4after HFS did notenhance the suppression of L-LTP, the fEPSPs still being152.2±6.6%and150.7±4.2%at the same time points (p>0.05)。(4) All drugs used in the experimentdid not affect PPF (P>0.05), which suggests that the inhibition of L-LTP inductionby ApoE4may be not mediated by presynaptic neurotransmitter release.These results indicated that apoE4injection before HFSs, not after HFSs,impaired hippocampal L-LTP, suggesting that neurotoxicity of ApoE4is mainlyinvolved in the suppression of L-LTP induction, but not the maintenance. In addition,the unaffected PPF suggests that the inhibition of L-LTP induction by ApoE4maymediated by postsynaptic signaling.Part II The Molecular Mechanisms Underlying the Neurotoxicity ofApoE4In order to clarify the molecular mechanism underlying the apoE4-inducedimpairment in the L-LTP induction, immunohistochemistry and Western Blottingwere used to find out whether the expression and phosphorylation levels of theCaMKII and CREB were changed or not. After the electrophysiological experiment,the brains in the same rats were used.The results showed that:(1)apoE4(0.2μg) injection30min before HFSs didnot affect the staining levels of CaMKII and CREB proteins inimmunohistochemistry experiments with the optical density of0.241±0.002and0.249±0.004, respectively (p>0.05). However, the levels of phosphorylatedCaMKIIα (Thr286) and CREB (Ser133) significantly decreased (p<0.01) afterapoE4injection, from0.250±0.008and0.275±0.002in control to0.094±0.002and0.170±0.002in apoE4group, respectively. Quantitative analysis with westernblotting also confirmed the findings above. The protein levels of phosphorylated CaMKIIα (Thr286) and CREB (Ser133) in apoE4(0.2μg) group significantlydecreased from the control of100%to61.74±5.36%and64.80±12.58%,respectively (p<0.05), while the values of CaMKII and CREB did not change, being97.25±4.57%and95.24±11.45%(p>0.05), respectively.(2) apoE4injection afterHFSs did not change the expression of phosphorylated CaMKIIα (Thr286) andCREB (Ser133), as well as CaMKII and CREB. The optical density ofphosphorylated CaMKIIα (Thr286) and CREB (Ser133) in apoE4(0.2μg) injection1min after HFSs was0.231±0.011and0.274±0.003, respectively, without anysignificant difference compared to the value in control group (p>0.05). Afterapplication of higher concentration of apoE4(2μg), the optical density was still0.228±0.003and0.278±0.003, respectively (p>0.05). Similar to the results ofimmunohistochemistry staining above, western blots quantification showed thatapoE4(0.2μg and2μg,) injection after HFSs did not change the expression ofCaMKII, CREB, phosphorylated CaMKII and CREB in the hippocampus (p>0.05).These results demonstrated that apoE4could impair hippocampal L-LTP byreducing p-CaMKIIα and p-CREB, and both CaMKIIα and CREB are importantintracellular targets of the neurotoxic apoE4.In conclusion, the present study, using field potential recording,immunohistochemistry and Western Blotting observed the effects of apoE4on thehippocampal L-LTP in vivo and the expression and phosphorylation levels ofCaMKIIα and CREB. These results indicated that neurotoxicity of apoE4is mainlyinvolved in the suppression of L-LTP. And the impairment may be mediated by thepostsynaptic signaling. In addition, both CaMKIIα and CREB are the importantintracellular targets. Therefore, the present experiment provides furtherelectrophysiological and molecular evidence for the neurotoxic effect of apoE4,which can contribute to the prevention and treatment of AD in the future.