| BackgroundThe progressive cognitive decline in Alzheimer’s disease(AD)patients,which is strongly age-associated,correlates with the extent of tau pathology.Hyperphosphorylation is the most important pathological change of tau protein.Although elevation of phosphorylated tau(p-Tau)on residues Thr181(p-Tau181),Thr217(p-Tau217),and Thr231(p-Tau181)in cerebrospinal fluid or blood are recently proposed to be particularly sensitive markers of early AD,the generation of p-Tau during brain activity is poorly understood.Long-term depression(LTD)and long-term potentiation(LTP),as two main forms of synaptic plasticity,are recognized as the biological basis of learning and memory.A few studies indicate that LTD induction enhances tau phosphorylation at Ser396(p-Tau396)and Ser202/Thr205(p-Tau202/205)in hippocampus in vitro.It is still unknown whether the expression levels of p-Tau181,p-Tau217 and p-Tau231 can also be enhanced by physiological LTD induction.Whether or not their enhancement is more sensitive compared with other reported residues.Whether or not the expression level of p-Tau can be affected by LTP induction.In addition,p-Tau in neurons at the isolated level has a shorter half-life and faster turnover rate,so the investigation of p-Tau changes in vivo can mimic clinic conditions better.Previous studies by our group have shown that combined injection of N-methylD-aspartate receptor(NMDAR)and metabotropic glutamate receptor subtype 5(m Glu R5)antagonists can effectively block the induction of LTD.Whether activation of these two receptors mediates LTD-triggered changes in p-Tau expression levels is unclear.It has been reported the level of integrated stress response(ISR)is aberrantly elevated in aged mice brain.A small molecule ISR inhibitor ISRIB(trans-isomer)reverses the aberrantly elevated ISR in aged mice brain and restores age-related changes in hippocampal neurons.In this research,hippocampal LTD and LTP will be induced in live Sprague Dawley(SD)rats of three different age groups(2-3 months,9-10 months and 17-18months).The effects of LTD or LTP on the expression level of p-Tau at different residues will be detected by immunofluorescence and Western blot techniques.The results of this research will provide a valuable in vivo means to(1)directly study the generation of p-Tau during learning and memory-related synaptic plasticity,and(2)aid in the selection of p-Tau directed therapies in future clinical trials of AD.Methods1 In vivo rat hippocampal LTD and LTP models established by in vivo electrophysiology.(1)SD rats of different months(2-3 months,9-10 months and 17-18 months)were taken and anesthetized by intraperitoneal injection of urethane,after recording the excitatory postsynaptic potential(EPSP)of the hippocampal CA3-CA1 neural pathway for 60 minutes,applying low frequency stimulation(LFS)to induce in vivo hippocampal LTD to construct in vivo hippocampal LTD models in rats of different months of age;(2)SD rats of different months(2-3 months,9-10 months and 17-18 months)were taken and anesthetized with urethane,after 60 minutes of recording EPSP in the CA3-CA1 pathway,applying high frequency stimulation(HFS)to induce in vivo hippocampal LTP to construct in vivo hippocampal LTP models in rats of different months of age.2.The expression levels of p-Tau at different sites were detected by immunofluorescence and Western blot.(1)Record 30 min after induction of LTD or LTP(Method 1).The expression levels of p-Tau181,p-Tau217,p-Tau231,p-Tau202/205 and p-Tau396 were detected by immunofluorescence.To determine the effects of LTD and LTP on the phosphorylation of the above sites of tau protein in the hippocampus of rats of different months of age;(2)Record 30 min after induction of LTD.The expression levels of p-Tau181,p-Tau217,p-Tau231,p-Tau202/205 and p-Tau396 in rat hippocampus were detected by Western blot.To further determine the effect of LTD in the hippocampus of rats of different months on the phosphorylation of the above sites of tau protein.3 Further clarification of the glutamate receptor mechanism of tau phosphorylation triggered by LTD in the rat hippocampus by pharmacological methods.(1)SD rats aged 17-18 months were anesthetized with urethane,intraperitoneal injection of NMDAR antagonist CPP(10 mg/kg)or m Glu R5 antagonist MTEP(3mg/kg)60 minutes before LFS,stable recording of CA3-CA1 neural pathway EPSP for60 minutes,applying LFS to induce LTD,and continuing to record EPSP for 30 minutes to confirm the induction effect of LTD.Then,the rats were perfused to take the brains for immunofluorescence staining,and the expression levels of p-Tau181 and p-Tau217 were detected respectively.To determine the type of glutamate receptors upon which LTD triggers tau phosphorylation in the hippocampus.4 To clarify the effect of ISRIB on the expression of p-Tau181 and p-Tau217 triggered by LTD in the hippocampus in vivo by pharmacological methods.(1)The prepared ISRIB solution was intraperitoneally injected(2.5 mg/kg)into SD rats.Plasma drug concentrations of ISRIB were measured by high performance liquid chromatography,to determine the pharmacokinetic profile of ISRIB in rats;(2)SD rats aged 17-18 months were treated with ISRIB(2.5 mg/kg)for 3 consecutive days,a single intraperitoneal injection per day,and then normal caged for 18 days after the treatment.Afterwards,in vivo electrophysiological experiments were performed,and recording was continued for 30 minutes after applying LFS to confirm the induction effect of LTD.The brains of rats were perfused for immunofluorescence staining,and the expression levels of p-Tau181 and p-Tau217 were detected respectively.To clarify the effect of LTD on the expression levels of p-Tau181 and pTau217 in the hippocampus of aged rats after ISRIB treatment.Results1.Induction of LTD enhances p-Tau181 and p-Tau217 in an age-dependent manner in live rats.In SD rats of different months(2-3 months,9-10 months and 17-18 months),stable LTD was induced by applying LFS(P < 0.05),and there was no significant difference in the amplitude of LTD among groups of animals of different ages.After electrophysiology,brain tissue was collected to detect the expression levels of p-Tau181,p-Tau217,p-Tau231,p-Tau202/205 and p-Tau396.We discover: in 2-3month old rats,the expression levels of p-Tau at the above sites did not change significantly;in 9-10 month old rats,except for the slight increase in the expression level of p-Tau217 in hippocampal CA1 area(P < 0.05),the expression levels of p-Tau at other detected sites did not change significantly;in 17-18 month old rats,the expression levels of p-Tau181 and p-Tau217 in whole dorsal hippocampus,CA1,CA3 and DG areas were significantly increased(P < 0.05),while the expression levels of pTau231,p-Tau202/205 and p-Tau396 did not change significantly.In order to further verify the above experimental results,another batch of aged SD rats was taken to detect the expression of p-Tau protein by Western blot after electrophysiology.The experimental results were consistent with the results of immunofluorescence,after LTD induction,the expression levels of p-Tau181 and pTau217 in the hippocampus were significantly increased(P < 0.05),but did not affect the expression of p-Tau at other sites.2.Blockage of NMDAR or m Glu R5 inhibits the elevation of both p-Tau181 and p-Tau217 triggered by LTD.Treated SD rats aged 17-18 months with CPP and MTEP,respectively,LFS induced stable LTD in both groups of rats(P < 0.05),and there was no significant difference between the two groups.After electrophysiology,the hippocampal brain tissue was taken to detect the expression of p-Tau181 and p-Tau217.In the CPP-injected aged rat group,the expression levels of p-Tau181 and p-Tau217 in the hippocampal CA1 area were not significantly changed except for the slight increase of p-Tau181 in the hippocampal CA1 area(P < 0.05);in the MTEP-injected aged rat group,the expression levels of pTau181 and p-Tau217 in different areas of the hippocampus did not change significantly.3.Inhibition of ISR blocks the elevated expression of p-Tau181 and p-Tau217 induced by LTD in the hippocampus of aged rats.In ISRIB-treated SD rats aged 17-18 months,administration of LFS induced stable in vivo hippocampal LTD(P < 0.05).After electrophysiology,the hippocampal brain tissue was taken to detect the expression of p-Tau181 and p-Tau217.We found that hippocampal LTD failed to significantly increase the expression levels of p-Tau181 and p-Tau217 in ISRIB-treated aged rats.4.LTP dose not cause changes in the expression levels of p-Tau(p-Tau181,pTau217,p-Tau231,p-Tau202/205 and p-Tau396).In SD rats of different months(2-3 months,9-10 months and 17-18 months),stable LTP was induced by applying HFS(P < 0.05),and there was no significant difference in the amplitude of LTP among groups of animals of different ages.The results of immunofluorescence showed that the expression levels of p-Tau(p-Tau181,p-Tau217,p-Tau231,p-Tau202/205 and p-Tau396)in the three age groups did not change significantly.Conclusions1.LTD triggers increased expression levels of p-Tau181 and p-Tau217 in rat hippocampus in an age-dependent manner;2.LTD-triggered elevated p-Tau181 and p-Tau217 expression levels in aged rats require co-activation of NMDAR and m Glu R5;3.Inhibition of ISR completely blocks LTD-triggered increases in p-Tau181 and pTau217 expression. |
PDF Full Text Request