Region-specific Expression Of Alzheimer-related Proteins And The Alterations Under ER Stress In Rats

Background:Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized clinically by progressive cognitive deficit. Intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques (SP) are due to accumulation of microtubule-associated protein tau and amyloid-β (AP), which are the main pathological characteristic accompanied by synaptic loss. There are selective that the NFTs and SP affect areas of brain in the process of AD and extend along with the disease progression. For example, earliest NFTs are observed in the transentorhinal and entorhinal cortex (Stage I and II). A more extensive involvement of the entorhinal cortex and the formation of NFTs in sector CA1of the hippocampus correspond to Stage Ⅲ and IV. In Stages V and VI abundant NFTs are presented in neocortical association areas. SP deposits are generally first found in neocortex, then the temporal lobe, finally appears in hippocampus and so on. The severity of the brain synaptic loss in AD patients is highly related to the number of NFTs in the same brain regions. The synaptic proteins are closely related to the degree of clinical dementia and hippocampus is the most severe lesion area where the synaptic lose, NFTs and SP form. Cerebellum is the lightest and latest lesion region in the procession of AD which causes our attention. Glycogen synthase kinase-3beta (GSK-3β) and protein phosphatase2A (PP2A) are important kinase and phosphatase which can regulate tau protein phosphorylation. Amyloid-β (Aβ) is the breakdown products of amyloid precursor protein (APP) under the action of β-and y-secretases. Endoplasmic reticulum (ER) stress is closely related to AD which can induce the phosphorylation of tau protein and deposion of Aβ. Therefore, this study further studied tau protein and its phosphorylation level, Aβ, synaptic related proteins in different brain regions and related molecular changes under ER stress.Aim:Compared with the level of total tau protein and phosphorylation tau at specific sites, APP, Aβ, synaptic related protein in different brain regions [temporal cortex (TC), frontal cortex (FC), hippocampus (HP) and cerebellum (CB)] of normal SD rats. Meanwhile, to observe the changes of those molecular under ER stress and analysis the possible mechanism.Methods:In this study, western blotting (WB), immunohistochemical (IHC) which can combine with specific antibodies were used to detect and analyze the level and distribution of certain proteins in different brain regions of normal male SD rats (4months old,280±20g, WB=6, IHC=3). These proteins include total tau, phosphorylated tau protein at specific sites, non phosphorylated tau protein, APP, phosphorylated APP, synaptic related protein [glutamate receptors1(GluRl), GluR2, N-methyl-D-aspartic receptors1(NR1), NR2A, NR2B, synapsin1and postsynaptic density protein95(PSD95)], glycogen synthase kinase-3β (GSK-3β), protein phosphatases2A (PP2A), mitogen-actived proein kinases (MAPKs) and so on. Simultaneously, astrocytes and microglia labeled by glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule1(Ibal) also are observed. Enzyme-linked immunosorbent assay (ELIS A) was used to detect and analyze the level of Aβ40and Aβ42. Then treated these rats with TM10μl,50μM, obtain the sample after48h) for the left lateral ventricular infusion, the coordinate of AP-0.8, L-1.5, V-3.8-4.0(in mm from bregma and dura, flat skull) was selected according to the stereotaxic atlas of Fanklin and Paxinos.Results:By western blotting, we observed the level of total tau did not show significant difference among the TC, FC, HP, CB of normal SD rats. And with TM injection48hours later, the level of total tau had no significant change in the four regions. The phosphorylated tau at Ser198/199/202, Thr205, Ser214and Ser262also showed no significant difference among the four regions. After TM being treated, the phosphorylated tau at Ser198/199/202increased significantly in TC, FC and HP but not in CB; at Thr205, Ser214sites increased in TC and FC but not in HP and CB; at Ser262site increased only in HP.The levels of phosphorylated tau at Ser404and Ser422sites in TC and FC were significantly higher than that in CB and HP of normal rats. And with TM being injedted48hours later, the level of them increased obviously in TC and FC but not in CB and HP. The levels of phosphorylated tau at Ser396, Thr231and Ser202/Thr205sites in CB were much lower than that in TC, FC and CB. And with TM being injedted48hours later, the level of phosphorylated tau at Ser396and Thr231sites had no changes in CB; at Ser396site increased significantly in TC and FC; at Thr231site increased significantly in FC and HP; while at Ser202/Thr205sites increased significantly in the four brain regions.The levels of total APP had no difference in TC, FC, CB and HP of normal rats and phosphorylated APP (p-APP) was much lower in CB and HP than that in TC and FC, and the level of p-APP in FC was much lower than that in FC. Furthermore, the level at the band of-72KDa molecular weight was much lower than that in TC, FC and CB. The level of Aβ40and the ratio of Aβ40/Aβ42had no difference in the four brain regions and the level of Aβ42in CB was much lower than that in TC and FC.4G8, the17aggregation of Aβ, was much lower in CB and HP than that in TC and FC. And with TM being injedted48hours later, the level of Aβ40, Aβ42and the ratio of Aβ40/AP42all had no changes in the four brain regions.The levels of GluR2, NR1, NR2A, NR2B and PSD95in CB were much lower than that in TC, FC and HP of normal rats; synapsin1in CB was much lower than that in TC; GluRl in CB and HP were much higher than that in TC and FC.The levels of protein kinase-like endoplasmic reticulum kinase (PERK) and inositol requiring1(IRE1) had no difference in the four brain regions and the level of activating transcription factor6(ATF6) in CB was much lower than that in the other three brain regions while the level of Bip in CB was much higher than that in TC, FC and HP. After treatment with TM, significant ER stress, demonstrated by the increased levels of p-PERK, p-IREl, p-ATF6and Bip, was induced in TC, FC and HP but not in CB. The similar results were observed with Bip and p-IREl by immunohistochemical.The level of GSK-3β in CB was much lower than that in TC, FC and HP; the phosphorylated GSK-3β at Ser9and Tyr216sites had no difference in the four brain regions; the phosphorylated PP2A at Tyr307site (Y307-PP2Ac) and demethylated PP2Ac (DM-PP2Ac) in CB and HP were much lower than that in TC and FC; extracelluar-signal regulated protein kinase (ERK), C-Jun amino terminal kinase (JNK), P38and p-P38had no difference in the four brain regions; p-ERK in CB was much lower than that in the other three regions.48hours after TM being injected, GSK-3β was activated in TC, FC and HP but not in CB; the level of total PP2A, Y307-PP2Ac, DM-PP2Ac had no changes in the four brain regions; p-ERK decreased significantly in TC, FC and HP but not in CB; p-JNK increased significantly in TC and FC but not in CB and HP; p-P38had no changes in the four brain regions.When exposure to TM, astrocyte dyed darker in TC, FC, HP and CA1, CA3, CA4, DG of HP with the cell body enlarged significantly and the number of cell of the other brain regions increased obviously except for DG. The results of microglia were similar to astrocyte, but the number of cell only increased in DG and the cell body enlarged in all the brain regions.Conclusion:Low level expression of the phosphorylated tau and no sensitive to TM may partially explain why cerebellum is often exempted from neurofibrillary tangles and SP. Low levels of GluR2, NR1, NR2A, NR2B and PSD95in cerebellum may illustrate why cerebellum only plays a very limited role in learning and memory. ER strees induced by TM can activate astrocyte and microglia. When ER stress was induced by TM, the phosphorylated tau at Ser396and Thr231sites increased significantly and synaptic related protein had no changes while the phosphorylated tau at multi-sites in HP increased significantly and synaptic related protein decreased obviously. The mechanism may involve in the increasing of ER stress related markers and the activation of GSK-3β，JNK and so on. Background:When intracellular excessive phosphorylated tau proteins form and extracellular Aβ aggregates in the early stage AD, the number of synapses has been significantly reduced. Synapses were the main structure to establish information communication among neurons. Combining with electron microscope, immune cell chemistry and biochemistry techniques, the researchers found that25-35%synapses lose in TC and FC after AD happened2to4years by brain tissue biopsy and the average loss synapse of each neuron was15-35%. Hippocampus synapses decreased most significantly with about44-55%of the synaptic loss and the synapses in TC lose lightest. More and more synapses lost along with the progression of AD. Further study found that the number of the synaptic loss was highly positive correlation to the severity of tangles in the same brain regions. About the results of relationship between synaptic loss and the number of SP were not consistent. Except for the decreasing number of synapses in the brain of AD, the decreasing synaptic proteins (including presynaptic protein, postsynaptic protein, synaptic vesicle proteins) are closely related to the degree of clinical dementia. In the part I experiment we observed the synaptic proteins in HP decreased significantly and Fu Zheng-Qi, one member of our team, ever observed that TM (10μl,50mM, lateral ventricle injection,48h) could induce spatial memory disorders of rats. Then what more behavior changes could be induced by TM, an ER stress revulant, after injected in lateral ventricle and what may be the possible molecular basis? This experiment will answer the two questions.Aim:To study whether the TM-induced ER stress causes memory deficits, spontaneous activity, anxiety and depression behavior change in rats by electric platform test, open field test, forced swimming test and high plus maze test and the related molecular mechanisms. Simultaneously, detected the signaling molecules related CREB to investigate the possible molecular mechanisms.Methods:In this study, TM was injected in the left lateral ventricle and the model replication was same to the part I, after24and48hours later, behavior tests were detected by electric platform test, open field test, forced swimming test and high plus maze test. Then the number of neuron, the number and type of dendritic spines, the level of glutamate receptors and synaptic related proteins and CREB associated signaling molecules in TC, FC, CB and HP were detect and analyze through Nissl staining, Golgi staining and Western blotting.Results:By electric platform test, we observed that the spatial memory disorders of rats rats could be induced by TM injected24and48hours later. By open field test, the spontaneous activity increased; by high plus maze, the time and number in the open arms were significantly higher than the control group; and by forced swimming test, there was no difference between TM group and control group. The behavioral results with early results of our team suggested that rats had the manifestation of memory disorders and increasing exploratory behaviour. In order to further study the mechanism of spatial memory impairment in rats induced by TM, we found the number of neurons in CA1decreased14%when exposure to TM but not in DG, CA3and TC, FC, CB by Nissl staining; the number of dendritic spines in DG reduced but not in CA1and CA3, the mushroom spine in CA1, CA3 and DG decreased while the thin spines reduced in CA1but increased in CA3and DG by Golgi staining.By western blotting, we observed the levels of GluR2, PSD95and synapsin1in HP decreased significantly when exposure to TM. In the part I test, we found GSK-3β being activated as well as the decreasing of p-ERK. In this part, we observed the level of PP1decreased and the cell cycle protein dependent kinase5(Cdk5) labeled by p25/35increased, the level of phosphorylated CREB at Ser133site decreased while at Ser129site increased.Conclusion:TM could induced memory disorders which was related to the reduce of number of neurons in CA1of HP, the decreasing of dendritic spines in DG decreasing, the decreasing of mushroom spine in HP and synaptic related proteins (GluR2, synpsinl, PSD95) reduce. The molecular mechanism may involve Cdk5, ERK/CREB or GSK-3β/CREB, etc.