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Background A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress, neuroinflammation, and abnormal mitochondrial function. In addition, iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease (AD). The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD. Objective To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma (SH-SY5Y) cells and tau hyperphosphorylation and aggregation. Methods SH-SY5Y cells were treated with ferric chloride (FeCl3) at four concentrations (10, 100, 200, and 400 mg·L−1). Cell survival rate was then detected by CCK8 assay. Intracellular iron content was determined prussian blue (Perl's) by iron staining after 24 h exposure to FeCl3 at 10 or 200 mg·L−1. Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression. The differences in the expression of the phosphorylated tau (p-tau) protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl3 at 10 and 200 mg·L−1. After dilution with phosphate buffered saline (PBS), FeCl3, human tauR3, and FeCl3 + tauR3 were incubated at 37℃, and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT) method at 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. Meanwhile, after 96 h coincubation of FeCl3 and tauR3, the fibers formed by tau aggregation were observed under a transmission electron microscope (TEM). Results After 24 h of FeCl3 exposure, the cell survival rate of SH-SY5Y cells among all groups was statistically different (F=8.63, P<0.01). The cell survival rates in the 200 and 400 mg·L−1 groups were 80.1% and 68.7% of the control group, respectively (P<0.05). Compared with the control group, the nuclei of the 200 mg·L−1 FeCl3 group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9% (P<0.01). After 24 h of FeCl3 exposure , the p-tau (Ser396) protein expression was statistically different among all groups (F=11.6, P<0.01). Compared with the control group, the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L−1 group was up-regulated by 72.7% (P<0.01). After FeCl3-treated SH-SY5Y cells with tau overexpression for 24 h, the p-tau (Ser396) protein expression was statistically different among all groups (F=27.8, P<0.01). Compared with the tau group, the p-tau (Ser396) protein expression level of SH-SY5Y cells in the tau + 200 mg·L−1 group was up-regulated by 44.6% (P<0.05). Compared with the tauR3 group, the fluorescence intensities in the 84 and 96 h tauR3 + FeCl3 groups were up-regulated by 49.9% and 53.7% (P<0.01) respectively. After 96 h of coincubation, compared with the tauR3 group, FeCl3 + tauR3 aggravated tau aggregation and formed fiber deposition under TEM. Conclusion Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability, promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid, and aggravate tauR3 aggregation and fiber production.
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Alzheimer's disease （AD） is a neurodegenerative disease. With the acceleration of aging process， the number of AD patients increases year by year. This threatens the health and even life of patients， and causes heavy economic burden and mental pressure to patients， families and society. In traditional Chinese medicine （TCM）， AD belongs to the category of dementia， and tonifying kidney is the main treatment. Based on the basic theory of TCM and combined with clinical manifestations of AD， AD is closely correlated with liver and spleen. Therefore， "simultaneous regulation of three Yin" of liver， spleen and kidney will be an important way for the prevention and treatment of AD. Hei Xiaoyaosan， a representative prescription of "simultaneous regulation of three Yin" of liver， spleen and kidney， has theoretical， experimental and clinical basis in preventing and treating AD. Modern studies have shown that neurofibrillary tangle formed by tau hyperphosphorylation is a main pathological feature of AD， and trimethylamine oxide （TMAO） is closely related to tau hyperphosphorylation. Therefore， regulating TMAO metabolism to inhibit tau hyperphosphorylation is a new target for the prevention and treatment of AD. On the basis of the above theory and previous studies， this paper put forward the hypothesis that Hei Xiaoyaosan regulates the trimethylamine（TMA）/heparin monooxygenase 3（FMO3）/TMAO metabolic pathway of intestinal flora through "simultaneous regulation of three Yin" of liver， spleen and kidney， and then inhibits tau hyperphosphorylation in brain hippocampus， thereby protecting nerve cells， improving learning and memory， and preventing AD. This paper explored the role and mechanism of Hei Xiaoyaosan in the prevention and treatment of AD from the perspective of inhibiting tau hyperphosphorylation by regulating the TMA/FMO3/TMAO metabolic pathway of intestinal flora， which provided new ideas and strategies for in-depth study of Hei Xiaoyaosan in the prevention and treatment of AD.
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Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, which is characterized by cognitive and memory dysfunction. Calpain is widely activated in cells. Disturbance of calpain is currently thought to a main cause of hyperphosphorylation and abnormal cleavage of tau protein in AD pathology. Calpain affects the activities of glycogen synthase kinase 3 and protein phosphatase 2A, which causes abnormal hyperphosphorylation of multiple sites of tau protein, and mediates truncation of tau protein monomers, and induces neurodegeneration. Calpain is expected to be a potential target for drug therapy of AD.
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Objective To study the role and mechanism of A2AR activation in tau hyperphosphorylation after brain injury.Methods SD rats were cultured with no specific pathogen level.SH-SY5Y was cultured.The rats were treated with CGS21680 solution and DMSO and SH-SY5Y respectively.The CGS21680 solution and sb216763, H-89, or Only add ZM241385, the control group plus DMSO, compared with each group tau hyperphosphorylation.Results The phosphorylation level of tau protein in SH-SY5Y cells was significantly higher than that in the control group (P<0.05).The phosphorylation level of tau protein in the primary hippocampal neurons of rats was significantly higher than that of the control group (P<0.05).The levels of tau protein phosphorylation in group 2 and group 3 were significantly higher than those in control group (P<0.05).The expression of tau in group 4 and group 5 was statistically significant (P<0.05)There was no significant difference in phosphorylation level between the two groups.Conclusion A2AR activation can activate kinase A and GSK-3β after brain injury, leading to tau hyperphosphorylation.
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Objective To investigate the effect of melatonin(MT)on tau hyperphosphorylation in hippocampus of mice with exposure to lead(Pb)at early development stage and its possible mechanism.Methods Healthy C57BL/6 mice were randomly divided into four groups:control group(received normal water),lead exposure group(exposed to 0.2% Pb acetate from postnatal day 1(PND 1)to PND 21 through drinking water),MT group(received 50 mg/mL MT through drinking water since 12-month-old for 3 months)and MT combined with lead exposure group(exposed to 0.2% Pb acetate from PND 1 to PND 21 and then given 50 mg/mL MT since 12-month-old for 3 months through drinking water).The Pb levels in the blood and hippocampus were determined by graphite furnace atomic absorption spectrometry.Morris water maze was used to detect the spatial learning and memory.The phosphorylation level of tau and the protein level of GRP78 and CHOP in hippocampus were detected by Western blotting.Results Compared with the control group,the Pb levels in the blood and hippocampus were significantly increased in lead exposure group(P<0.05),while MT treatment did not affect the Pb levels both in the blood and hippocampus.In the lead exposure group,the phosphorylation level of tau in the hippocampus was significantly increased compared with control group(P<0.01),and after treatment with MT,the phosphorylation level of tau in MT combined with lead exposure group was significantly decreased compared with the lead exposure group(P<0.05).Furthermore,the expression of GRP78 and CHOP in the hippocampus was significantly higher in lead exposure group than in control group(P<0.05),and after treatment with MT,the expression levels of GRP78 and CHOP were lower than those of the control group.Conclusion MT can ameliorate the phosphorylation level of tau in the hippocampus and defects of learning and memory in C57BL/6 mice with exposure to lead at early development stage,and this may be related with the modulation of endoplasmic reticulum stress.
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Aim To research the synergistic effect of hyperlipoproteinemia and Aβ in the processing of Alzheimer′s disease.Methods Seventy SD rats were randomly divided into seven groups, and dealt with D-gal(hypodermic injection), hyperlipemia diet, microinjection into both side of CA1 section in hippocampus, independently.Morris water maze(MWM) test was used to evaluate the spatial memory impairments.Tau and tau(pThr181) pathology in the hippocampus were detected using Western blot and immunohistochemistry.Nissl′s staining was used to detect cell apoptosis.Results Aβ25-35-treated rats showed significant impairments of spatial memory in MWM test, especially in the group of D-gal+Aβ25-35+HLD(P<0.01).Furthermore, these rats treated with Aβ25-35, D-gal, and hyperlipemia diet, exhibited significantly increased phosphorylation of tau, particularly in the Thr181 site.Conclusion Hyperlipoproteinemia is the risk factor for older person, which could strengthen the toxic effect of Aβ, and promote phosphorylation of tau.
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OBJECTIVE: To investigate the regulating effects and its possible mechanism of safflower yellow (SY) on tau protein hyperphosphorylation in SH-SY 5Y cell induced by okadaic acid (OA). METHODS: Cell viability was measured by MTT colorimetric method. The cell apoptosis was determined with Hoechst 33342 after SH-SY 5Y cell were treated with SY. To determined the phos-phorylated tau protein of p-tau (T205), p-tau(S199) sites, and total GSK-3β, PP2A in SH-SY 5Y cell were determined using Western blotting. RESULTS: SY significantly increased survival rates of SH-SY5Y cell damaged by OA. Hoechst 33342 showed that SY decreased the apoptosis of SH-SY5Y cells. Western blotting analysis showed that tau protein content was increased compared to control group at p-tau (T205), p-tau (S199) sites. However, SY treatment significantly reduced tau protein content at p-tau (T205) and p-tau (S199) sites. PP2A content was significantly suppressed by OA, significantly increased by SY, GSK-3β content was significantly increased by OA and significantly decreased by SY. CONCLUSION: These results suggest that safflower yellow may have neuroprotective effect on OA-induced SH-SY 5Y cells injury, which may be associated with tau hyperphosphorylation at the enzyme level by activation of tau kinases or by a decline in the activities of tau phosphatases.
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<p><b>OBJECTIVE</b>To study the effect of chronic noise exposure on expression of N-methyl-D-aspartic acid receptor 2B (NR2B) and tau phosphorylation in hippocampus of rats.</p><p><b>METHODS</b>Twenty-four male SD rats were divided in control group and chronic noise exposure group. NR2B expression and tau phosphorylation in hippocampus of rats were detected after chronic noise exposure (100 dB SPL white noise, 4 h/d×30d) and their mechanisms underlying neuronal apoptosis in hippocampus of rats with TUNEL staining.</p><p><b>RESULTS</b>The NR2B expression decreased significantly after chronic noise exposure which resulted in tau hyperphosphorylation and neural apoptosis in hippocampus of rats. Immunohistochemistry showed that the tau hyperphosphorylation was most prominent in dentate gyrus (DG) and CA1 region of rat hippocampus.</p><p><b>CONCLUSION</b>The abnormality of neurotransmitter system, especially Glu and NR2B containing NMDA receptor, and tau hyperphosphorylation in hippocampus of rats, may play a role in chronic noise-induced neural apoptosis and cognition impairment.</p>
Subject(s)
Animals , Male , Rats , Apoptosis , Hippocampus , Cell Biology , Metabolism , Neurons , Cell Biology , Metabolism , Noise , Phosphorylation , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate , Metabolism , tau Proteins , MetabolismABSTRACT
significantly attenuated the hyper-phosphorylation of tau proteins. These data suggested that inhibition of cdk-5 activity has neuropro-tective effect on neurons in NPC mice.
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AIM: To probe into tau hyperphosphorylation at PHF- 1 sites induced by glycogen synthase kinase - 3β(GSK- 3β) in vivo. METHODS: Twenty - one rats were randomly allocated to three groups as follows: GSK - 3β transfection group, vector group and control group; 0.1 μg/3μL GSK- 3β- HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK- 3β- HA plasmid and tau phosphorylation using phosphorylation- dependent tau antibody PHF- 1. RESULTS: After transfection with GSK- 3β- HA for 48 h, GSK - 3β - HA was expressed in GSK- 3β transfection group; and hyperphosphorylated tau at PHF- 1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK- 3β expressed by the transfected GSK- 3β plasmid. CONCLUSIONS: Transfection with GSK- 3βin vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK- 3β is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy- related neurodegenerative diseases.
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Objective To study the protective effects of estrogen on function of learning-memory and the neuron of hippocampus in the ovariectomized(OVX) rat model.Methods Estrogen 200 ?g/kg was injected into rats twice a weak followed the OVX rat models were estestablished(OVX+E2 group).The function of learning-memory was tested by Morris water maze;the tau hyperphosphorylation was detected by immunohistochemistry staining;the pathological changes of hippocampus was observed by HE staining and Bielschowski staining.Results Compared with OVX group,the function of learning-memory of Morris water maze in OVX+E2 group were significant improved(all P
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The purpose of the present work is to observe whether Tau protein Ser202/Thr205 is hyperphosphorylated in braintissues of diabetic mice and to study the effect of App17 peptide. Mouse diabetic model was produced with streptozotocin, andApp1 7 peptide as a treatment was injected subcutaneously into diabetic mice. Four weeks later, fixative was injected intravascu-larly into the mice, the brain was removed and crystat sections prepared. Immunohistochemical staining was done with AT-8. Inthe brains of diabetic mice positive AT-8 reacting neurons were numerous, darkly stained, and widely distributed in retrosplenialgranular cortex, hippocampus, thalamus et al. , while in normal mice and App17 peptide-treated diabetic mice positive cells werescarce and poorly stained. Tau protein is hyperphosphorylated at Scr202/Thr205 site and widely distributed in the brains of dia-betic mice, while App17 peptide can normalize the expression of AT-8 positive cells.
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Objective To explore the effect of hyperphosphorylated tau protein on the formation of brain amyloid.Methods Okadaic acid was injected into lateral ventricle of rats, once a day for eight weeks. The place navigation and spatial probe ability of rats were assessed by Morris water maze. Immunohistochemistry techniques were used to detect the expressions of neurofibrillary tangles and amyloid.Results In the Okadaic acid group, the spatial learning and memory abilities of rats were significantly impaired. The mean incubation period of Morris water maze was longer than control group. The accumulation of neurofibrillary tangles and plenty of ?-amyloid positive cells were detected in hippocampus CA1, CA3 and CA4 regions, dentate gyrus and cortex. Deposition of ?-amyloid was observed in hippocampus CA1 and CA3 regions, dentate gyrus and cortex.Conclusion The hyperphosphorylated tau protein may significantly increase deposition of amyloid in brain.
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AIM: To probe into tau hyperphosphorylation at PHF-1 sites induced by glycogen synthase kinase-3? (GSK-3?) in vivo. METHODS: Twenty-one rats were randomly allocated to three groups as follows: GSK-3? transfection group, vector group and control group; 0.1 ?g/3 ?L GSK-3?-HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK-3?-HA plasmid and tau phosphorylation using phosphorylation-dependent tau antibody PHF-1. RESULTS: After transfection with GSK-3?-HA for 48 h, GSK-3?-HA was expressed in GSK-3? transfection group; and hyperphosphorylated tau at PHF-1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK-3? expressed by the transfected GSK-3? plasmid. CONCLUSIONS: Transfection with GSK-3? in vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK-3? is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy-related neurodegenerative diseases.
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Tau is a multifunctional protein that are originally identified as a microtubule-associated protein. Tau is mainly expressed in neurons. There is a protein phosphorylation/dephosphorylation imbalance and tau is abnormally hyperphosphorylatied in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles(PHF/NFT). The modulation of the activities of phosphatase(PP)-2A, PP1 and tau kinase, such as GSK-3, cdk5 and MAPK effect tau protein hyperphosphorylation. It is unclear about the relation between tau and A? in AD, increased or altered A? production may be an initiating event in the pathogenic process in AD; but aberrant alternations in tau function are likely necessary for the subsequent neuronal dysfunction and death. Therefore, clearly defining the metabolism and function of tau in both normal and disease states is necessary for our understanding of AD and the development of appropriate therapeutic interventions.
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Objective To investigate the in vivo effect of melatonin(Mel) on calyculin A(CA)-induced tau hyperphosphorylation in neuroblastoma cells(N2awt).Methods We treated N2awt cells with CA or CA and 50 ?mol/L Mel,detected the level of tau phosphorylation with immunofluorescence,and assayed the activities of GSK3 and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3?.Results CA treatment led to tau hyperphosphorylation accompanied with the increased activity of GSK-3 and the decreased ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3?.When the cells were incubated simultaneously with CA and 50 ?mol/L Mel,the CA-induced tau hyperphosphorylation,GSK-3 activation and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3? decrease were attenuated.Conclusion Melatonin protects neuroblastoma cells from CA-induced tau hyperphosphorylation.Its protection may be related to the regulation of GSK-3 activity and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3? increase.
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Objective To investigate the in vivo effect of melatonin on calyculin A-induced neurofilament(NF) hyperphosphorylation in neuroblastma cells(N2awt).Methods N2awt cells were treated with CA or CA and different concentration melatonin or CA and vitamin E,the levels of neurofilament phosphorylation and the level of PP-2A were detected,and the activities of PP-2A were assayed.Results Calyculin A treatment led to neurofilament hyperphosphorylation by decreasing the level and activity of PP-2A.Both melatonin and vitamin E had protective effect on calyculin A-induced neurofilament hyperphosphorylation,although melatonin increased the activity of PP-2A while vitamin E did not.Morever,melatonin partially attenuated the decreasing of PP-2A level. Conclusion Melatonin protects neuroblastma cells from CA-induced neurofilament hyperphosphorylation through the regulation of PP-2A level and the increase of PP-2A activity.
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Objective Through the observation on the distribution of hyperphosphorylated Tau,to investigate the connection between hyperphosphorylated Tau and learning, memory tasks. Furthermore, the treatment of App17 on brain tissues of diabetic mice. Methods Diabetic model mouse was produced in the use of streptozotion and App17 peptide as a curative was injected subcutaneously. Four weeks later, removed the brains. Immunohistochemical stainning was done with AT\|8, Tau\|1, again with Tau\|1 antibody after dephosphorylation. Results In the brains of diabetic mice positive AT\|8 reacting neurons were widely distribution in retrosplenial granular cortex, hippocampas, thalamus et al, the cytoplasm was darkly stained, while in normal mice and App17 peptide\|treated diabetic mice positive cells were localized in retrosplenial granular cortex, however, in hippocampas and RSG area, the cytoplasm were poorly stained. Conclusion Hyperphosphorylated Tau is widely expressed in brains of diabetic mice. App17 peptide can improve the hyperphosphorylated Tau in brains of diabetic mice, therefore, it may improve learning ability and memory.\;