Effect Of14-3-3on SET Nuclear Transportation And Its Molecular Mechanism In Alzheimer Disease

Background:Protein phosphatase-2A (PP2A) activity is believed to be a main cause of the abnormal hyperphosphorylation of tau and neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) brain. I2PP2A/SET is an endogenous and effective inhibitor of PP2A. The SET transcription and expression is increased and mainly distributed in the olfactory bulb, hippocampus, cerebral cortex, caudate putamen, thalamus and other brain areas in AD brain. As a nuclear protein, SET translocates from nuclei to the cytoplasm in the neurons, which localizes with both abnormally hyperphosphorylated tau and PP2A. Previous studies have found that overexpression of SET leads to a significant reduction PP2A activity, tau hyperphosphorylation, neuronal degeneration, and even the performance of motor dysfunction in rats like AD patients. The results indicate that as an upstream regulatory factors of PP2A, SET transports from nuclear and accumulates in the cytoplasm is a key factor causing the onset of AD. Thus, explaining SET specific nuclear transport mechanism will help interpret the pathogenesis of AD. However, the detailed regulatory mechanism by which SET is detained in cytosol and the consequent PP2A inactivation in mammalian cells is not fully elucidated. Our previous study showed that phosphorylation of Ser-9inactivates SET Nuclear localization signal (NLS), phosphorylated SET is hard to bind with importin proteins and accumulates in the cytoplasm. However, the molecular mechanism of SET Ser-9phosphorylation and other possible mechanisms of SET aggregation induced cytoplasmic retention need to be further clarified.14-3-3protein defined as a joint/scaffold protein, which extensively involved in apoptosis, cell division, regulation of signal transduction processes, membrane transport proteins and other important life events. Some studies have revealed that14-3-3proteins play a key role in the regulation of nuclear transport. Phosphorylated Cdc25(Ser-216) interacts with14-3-38to form a complex Cdc25/14-3-3s, which induces Cdc25binding with importina impossible to go back to nuclei, similar as RSG, HDAC4proteins et.al.. These studies suggest that14-3-3protein has a negative effect for the nuclear-cytoplasm shuttling protein. Previous studies have found that14-3-3ξ is overexpression in the brain of AD patients, and its expression levels positively correlated with AD process. These studies suggest that14-3-3p/^protein involved in the pathogenesis of AD. However, the underlying mechanism is unknown. As a negative adaptor of the nuclear-cytoplasm shuttling,14-3-3protein binds to the target protein with a consensus binding sequence. By scanning the full-length sequence of SET, we found some serine-rich sequence146-156aa NESGDPSSKST consistent with the binding sequence RX1-2SX2-3S (X instead of any essential amino acids). It implies that14-3-3protein may combine with SET and regulate its nuclear import process.Objective:To investigate whether overexpression of14-3-3β/ξ causing SET nuclear transport disorder and the key sequence of14-3-3binding with SET, and further elucidate the pathogenesis of AD and seek effective molecular targets for drug development.Methods:HEK293/tau cells were cultured. Confocal laser scanning microscopy (LSM) and Western blotting (WB) were used to detect the subcellular distribution of SET at48h after14-3-3β/ξ transfection. Detected whether SET binding with14-3-3β/ξ and importina by Co-immunoprecipitation method.14-3-3ξ transfected HEK293/tau cells added purified synthetic peptide Tat-NESGDPSSKST (Peptide1) and Tat-NSSTEDGPKSS (Peptide2) simultaneously,48h later, LSM and WB were used to detect the subcellular distribution of SET. Detected the degree of integration of SET and importina in Peptide1/2treated14-3-3ξgroups with Co-immunoprecipitation method. The PP2Aactivity Assay kit (Promega, Cat.#V2460) was used to determine PP2A activity. Western blotting was used to detect tau phosphorylation sites at Ser199, Ser202, Thr205, Thr231, Ser262, Ser396and Ser404. To further demonstrate NESGDPSSKST is the right sequence of SET binding with14-3-3, which can inhibit14-3-3binding with SET, play a key role in SET nuclear transport. Adeno-associated virus (AAV) packaging14-3-3ξ was used to infect C57mice brain. Peptides1/2were used to test the efficacy. Contextual fear condition, Morris water maze test were performed to detect the changes in behavior, LSM was used to verify SET subcellular localization in brain slices,64array system was used to detect the LTP changes in living brain slices of hippocampal from DG to CA1region, primary cultured rat embryonic hippocampal neurons were used to find the changes in neuronal dendrites, dendritic spines, WB was used to test the synaptotagmin, synaptophysin, synapsin1, phosphorylated synapsin1(p-synapsin1), NR1, NR2A, NR2B and SET Ser-9phosphorylation level in hippocampus tissue of mice.Results:In this study, we found that compared with the control group, Most SET in14-3-3β/ξ overexpressed groups transfer and retain in the cytoplasm in HEK293/tau cell lines. Furthermore, we confirmed it by isolated cytoplasmic and nuclear SET with a nuclear protein extraction kit. Overexpression of14-3-3mβ/ξ induced SET accumulation in cytoplasm in HEK293/tau cells, which were resulted from decreased importina binding. We also observed that compared with the control group, most of SET in HEK293/tau cells treated with Peptide1will go back to the nucleus. Furthermore, we confirmed this conclusion by isolated cytoplasmic and nuclear SET with a nuclear protein extraction kit. We found that SET level was significantly increased in the nucleus, while significantly reducing in the cytoplasm with a treatment of Peptide1, which improves SET nuclear import by interaction with importina. Overexpression of14-3-3β/ξ significantly inhibits PP2A activity and cause tau hyperphosphorylation at Ser199, Ser202, Thr205, Thr231, Ser262, Ser396and Ser404sites. Peptide1treatment restores PP2A activity. Adeno-associated virus (AAV) packaging overexpression of14-3-3ξ was used to infect C57mice at DG region. Peptides1/2were used to test the efficacy. Contextual fear condition, Morris water maze test were performed a learning and memory deficit, and Peptide1restores them. LSM was used to verify SET subcellular localization in different area of brain slices. We found that Peptide1treatment recovers SET accumulation in the cytoplasm induced by overexpression of14-3-3^.64array system was used to detect the LTP changes in brain slices of hippocampal from DG to CA1region, we observed that Peptide1treatment enhances the reduction of LTP caused by overexpression of14-3-3ξ. Primary cultured rat embryonic hippocampal neurons were used to find that neuronal dendrites length, dendritic branches and spine density reduced by overexpression of14-3-3ξ, while Peptide1recovers these changes. WB was used to test the synaptotagmin, synaptophysin, synapsin1, phosphorylated synapsin1(p-synapsin1), NR1, NR2A, NR2B and SET pSer-9level changes in hippocampus tissue of mice, we found that overexpressed14-3-3ξ leads synaptotagmin, synaptophysin, synapsin1, p-synapsin1level reduced, NR2A/NR2B ratio and SET pSer-9level increased, while Peptide1treatment effectively restored these alteration.Conclusion:14-3-3β/ξ binding the sequence NESGDPSSKST of SET leads to a reduction of the interaction between SET and importina, results in SET nuclear import dysfunction. The cytoplasmic aggregation SET significantly inhibits PP2A activity, and consequently causes hyperphosphorylation of tau, neuronal dendritic impairment, LTP and learning and memory damage, while treatment of Tat-NESGDPSSKST competitively combining with14-3-3β/ξ induces SET going back to nuclei, and restores PP2A activity, reduces neuronal dendritic impairment, recovers LTP and learning and memory deficits. Background:Our previous studies have found that overexpression of14-3-3^leads to SET retention in the cytoplasm, and promote SET phosphorylation at Ser-9. However, upregulation of CKII also increases SET Ser-9phosphorylation and enhance SET retention in the cytoplasm. In AD brain, CKII level is increased.14-3-3dimer can recruit CKII. We assume that14-3-3ξ combine with both SET and CKII, and then SET becomes more easy to be phosphorylated by CKII. Therefore, phosphorylated SET is hard to bind with importina, finally resulting in SET retention in the cytoplasm.Objective:To investigate whether14-3-3ξ interacts with CKII and explore the mechanism that how SET is detained in the cytoplasm, and further elucidate the pathogenesis of AD.Methods:Adeno-associated virus (AAV) packaging14-3-3ξ was used to infect C57mice. LSM was used to verify the binding between SET,14-3-3ξ, CKIIa, and SET subcellular localization in brain slices. Co-immunoprecipitation was performed to detect the binding between SET,14-3-3ξ, CKIIa48h after14-3-3β/ξ transfections respectively. The amount of SET pSer-9, SET, CKIIa were detected respectively after treatment with CKII specific activity inhibitor TBB in HEK293/tau cell transfected with14-3-3ξ. The amount of SET pSer-9, SET, CKIIa were detected respectively after treatment with si14-3-3ξ in HEK293/tau cell transfected with CKII. PP2A activity employed PP2A activity Assay kit (Promega, Cat.#V2460). AAV packaging14-3-3ξ was used to infect C57mice, and the14-3-3ξ mice were treated with AAV8-sil4-3-3ξ, AAV8-Ssi respectively. Contextual fear condition, Morris water maze test were used to detect the changes in behavior. LSM was used to verify SET subcellular localization in different area of brain slices.64array system was used to detect the LTP changes in living brain slices of hippocampal from DG to CA1region, primary cultured rat embryonic hippocampal neurons were used to find the changes in neuronal dendrites, dendritic spines, WB was used to test the synaptotagmin, synaptophysin, synapsin1, phosphorylated synapsin1(p-synapsin1), NR1, NR2A, NR2B and SET Ser-9phosphorylation level in hippocampus tissue of mice.Results:In this study, we found that SET,14-3-3ξ, CKIIa were interacted with each other when14-3-3ξ was overexpressed, and SET in14-3-3ξ mouse mainly localized in the cytoplasm. In HEK293/tau cell lines, co-immunoprecipitation showed that SET, CKIIa,14-3-3ξ were combined with each other, and the combination degree was significantly increased under overexpression of14-3-3ξ. We observed that inhibit the activity of CKII induced a decrease of SET pSer-9after treatment with CKII specific activity inhibitor TBB in HEK293/tau cell transfected with14-3-3ξ.We found that knocked down14-3-3ξ lead to reduced SET pSer-9after treatment with si14-3-3ξ in HEK293/tau cell transfected with CKII. TBB recovered PP2A activity. AAV8-CKIIa infected C57mice were treated with AAV8-sil4-3-3ξ,AAV8-Ssi respectively. Behavioral tests showed that knock down of14-3-3ξ restored learning and memory impairments. CKIIa leads to SET retention in the cytoplasm, while si14-3-3ξ promoted the SET nuclear importin.64array system was used to detect the LTP changes in brain slices of hippocampal from DG to CA1region. We observed that si14-3-3ξ treatment enhanced the reduction of LTP caused by overexpression of CKIIa. Primary cultured rat embryonic hippocampal neurons were used to detect neuronal dendrites length, dendritic branches and spine density reduced by overexpression of CKIIa, while si14-3-3ξ recovers these changes. WB was used to test the synaptotagmin, synaptophysin, synapsin1, phosphorylated synapsin1(p-synapsin1), NR1, NR2A, NR2B and SET pSer-9level changes in hippocampus tissue of mice, we found that overexpressed CKIIa leads synaptotagmin, synaptophysin, synapsin1, p-synapsin1level reduced, NR2A:NR2B ratio and SET pSer-9level increased, while si14-3-3ξ treatment effectively restored these alteration.Conclusion:14-3-3ξ interacts with both CKIIa and SET simultaneously, promotes CKII-catalyzed SET Ser-9phosphorylation, induces neuronal dendritic impairment, LTP and learning and memory damage. si14-3-3ξ reduces the combination of CKIIa and SET, causes a significant decreased CK2-catalyzed SET Ser-9phosphorylation, thus restores the activity of PP2A, reduces neuronal dendritic impairment, recovers LTP and learning and memory deficits.