| Backgroud:In 2010, the number of people suffering Alzheimer’s disease (AD) in China was 5.69 million. The total prevalence among people above 65 was about 3.5%. AD threatens the health of the elderly and reduces the quality of life of both patients and their families. The global cost for AD was about 600 billion dollars every year. This situation is challenging for the whole society and families with heavy burden on caring and treatment. Studying AD pathogesis has important significance. AD is an irreversible neurodegenerative disease characterized with amyloid-beta (Aβ) deposits, intracellular neurofibrillary tangles and neuronal loss. The main clinical manifestations of AD are loss of memory, cognitive disorders, behavioral impairment, mental disorders, and impaired daily living activities. The researches of AD pathogenesis have been focused on genetic factors, (3-amyloid cascade hypothesis, cholinergic hypothesis, inflammation, oxidative stress and endoplasmic reticulum stress (ERS), energy metabolism, insulin signaling impairment, excitotoxicity and vascular factors. Among these mechanisms, ERS and insulin signaling impairment attracted the most concern. In the adipose cells, it was proved that ERS could lead to insulin signaling impairment by the downstream kinase c-Jun NH2-terminal kinase (JNK)-mediated serine phosphorylation of insulin receptor substance-1 (IRS-1), casuing the insulin resistance of the adipose tissue. Similar results were obtained in liver and skeletal muscles. Little was known about their connections in AD. In AD, whether ERS could lead to insulin signaling impairment and the underlying mechanism still remain unclear.Endoplasmic reticulum is responsible for protein synthesis, processing and modification in cells. Unfolded or misfolded proteins accumulating in the endoplasmic reticulum lumen triggers ERS. When ERS occurs, the binding immunoglobulin protein (BiP) dissociates from inositol-requiring enzyme 1α (IRE1α), double strand RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6), inducing activation of downstream signals. In the early stage, ERS can alleviate the stress by increasing protein degradation, promoting the expression of molecular chaperones and decreasing protein synthesis. But if ERS sustains chronically or is too strong and prolonged, programmed cell apoptosis will be induced. As the post-mitotic cell, neurons will suffer more stress due to being incapable of replication and division. In human samples from autopsy of AD patients, ERS was observed. Expression of BiP was increased, which was associated with Ap deposition. Elevated PERK phosphorylation could be detected in the brain of AD patients with co-localization of Tau hyperphosphorylation. In the hippocampus and temporal lobe of AD patients, phosphorylation of IRE la and upregulated X-box binding protein 1(XBP1) mRNA splicing were detected. In Aβ oligomers treated primary neurons, activation of ERS could be observed. The activation of ERS was marked by increased expression of BiP and CEBP homology protein (CHOP), elevated phosphorylation of PERK and XBP1 mRNA splicing. ERS can activate the downstream kinase JNK, which plays an important role in the pathogenesis of AD. In Aβ oligomers treated neuron-like cells and primary neurons, JNK phosphorylation was increased. In the cortex and hippocampus of AD transgenic mice, increased phosphorylation of JNK could be detected. In the autopsy of AD patients, JNK phosphorylation was elevated with Aβ co-localization. Intraperitoneal injection of JNK inhibitors could improve the Morris water maze performance of AD transgenic animal. JNK3 knockout mice displayed reduction in Aβ1-42 levels and Aβ plaque load, increased amount of neurons and improved cognitive behavioral detection.Insulin exerts its biological effects by binding to a subunit of the tyrosinekinase insulin receptors and activating the tyrosine kinase activity of the (3 subunits, resulting in receptor autophosphorylation and subsequently phosphorylation of intracellular IRS-1 on tyrosine residues. Tyrosine phosphorylated IRS-1 recruiting phosphatidylinositol 3-kinase (PI3K) causes the downstream protein kinase B (PKB/ Akt) and glycogen synthase kinase 30 (GSK3β) activated. This process is the brief introduction of insulin PI3K/Akt signaling. In most studies, IRS-1 is the key molecule that mediates insulin signaling impairment. Most sites of IRS-1 serine phosphorylation can lead to IRS-1 disassociation from PI3K or insulin receptor, reduction of IRS-1 tyrosine phosphorylation, and interruption of insulin signaling. The complex balance between serine phosphorylation and tyrosine phosphorylation of IRS-1 mediated the insulin signaling status. Studies have shown that multiple kinases can lead to IRS-1 serine phosphorylation. JNK is the important one and responsible for IRS-1 serine phosphorylation of 307,318,612 sites. JNK might be the key molecular linking ERS and insulin signaling impairment.Insulin signaling is involved in the processes of learning and memory at an extremely important position. It has been proved that distribution of insulin receptor is at high density in cognitive sensitive regional of brain, such as the hippocampus, amygdala, and medial temporal lobe. Spatial memory training could increase the sensitivity of hippocampus insulin receptor obviously by changing expression, activity, translocation and tyrosine phosphorylation status. While specific blockade of the hippocampus insulin receptor could lead to memory disorders in experimental animals. Since Hoyer S first proposed insulin signaling impairment as the pathogenesis of AD in 1994, it has been gained extensive attention year by year. Insulin signaling impairment played a vital role in the AD pathogenesis, and was involved in Aβ generation and removal, neurogenesis, Tau hyperphosphorylation, energy metabolism and synaptic function. Increased IRS-1 serine phosphorylation at 307,612 sites were observed in Aβ treated primary neurons. In the transgenic mice 3×Tg, IRS-1 serine phosphorylation at 612 was increased. Increased serine phosphorylation of IRS-1 at 312,616,636 (equivalent to rodent 307,612,632) sites and changes of basal levels of phosphorylated Akt were proved in the autopsy of AD patients. It was repoted that increased serine phosphorylation of IRS-1 at 616 site was colocalized with intracellular neurofibrillary tangles. All above, IRS-1 serine phosphorylation was significant in insulin signaling impairment in AD.In AD pathogenisis, ERS was the proved early event. And ERS could lead to JNK activation. Moreover, JNK could mediate the serine phosphorylation of IRS-1. IRS-1 is the key molecule in insulin signaling, and its serine phosphorylation could lead to insulin signaling impairment. ERS, JNK activation and insulin signaling impairment were significant in AD pathogenesis. Besides, in the 9-month-old APP/PS1 mice, ERS, JNK activation and insulin signaling impairment were observed. Therefore, we hypothesized that ERS might mediate the insulin signaling impairment and its underlying mechanism might be JNK-dependent IRS-1 serine phosphorylation at 307,318,612 sites.Highly differentiated PC 12 cells were regarded as neuron-like cells to be used in AD research. For further research, cotical neurons from newly-born Wistar rats were employed. AP1-42 oligomers were the most toxic to neurons in AD brain. Aβ1-42 oligomers-treated neuron-like cells or neurons was the first choice for AD cell model. APP/PS1 mice carrying the human APPswe mutations (K594N/M595L) and presenilin-1 with the exon 9 deletion, are generally accepted as the mature model of Alzheimer’s disease. ERS inducer thapsigargin (TG), ERS inhibitor 4-phenyl butyric acid sodium (PBA) and JNK inhibitor SP600125 are generally accepted. And the PBA and SP600125 could cross the blood-brain barrier. To test our hypothesis, Aβ1-42 oligomers treated PC12 cells, cortical primary neurons and APP/PS1 mice were selected for our experiment, using ERS inducer, ERS inhibitor and JNK inhibitor.Aims:1. To confirm ERS and insulin signaling impairment in AD cell model and AD transgenic mice.2. To investigate whether ERS mediate insulin signaling impairment in AD cell model and AD transgenic mice.3. To clarify the underlying mechanism of ERS-mediated insulin signaling impairment in AD cell model and AD transgenic mice.Methods:1. In vitro experimentsCulture of highly differentiated neuron-like PC12 cells:PC 12 cells were obtained from Shanghai Institutes for Biological Sciences (Catalog number TCR9). PC 12 cells were maintained at 37℃ in an atmosphere of 5% CO2 in high glucose Dulbecco’s modified Eagle’s medium (DMEM), which contained 10% fetal bovine serum and 1% penicillin/streptomycin.Culture and identification of cortical primary neurons:Cortical neurons were deprived from Wistar newly born rat (Experimental Animal Center of Shandong University). Neurons were maintained at 37℃ in an atmosphere of 5% CO2 incubator in Neurobasal A medium in the presence of 2% serum-free B27,0.5% L-glutamine and 1% penicillin/streptomycin for 10 days. The indentification of neurons was carried out by immunofluorescence MAP2 and GFAP.2. In vivo experimentsFeeding and identifying of AD transgenic mice:AD transgenic mice and the corresponding wild type (WT) mice were obtained from Nanjing Model Animal Research Center of Nanjing University (from Jackson Laboratories). The strain of AD transgenic mice is B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/Mm JNju (APP/PS1 for short). All the experimental animals were kept in IVC system in SPF environment with constant temperature at 22 ± 3℃, relative humidity at about 60%, automatic light control mimicking normal circadian rhythm. All the procedures were carried out according to the standard of SPF level. The experimental mice were underwent genotype identification at 1-month-old by extracting DNA from the tips of tail. The 9-month-old mice were indentified by immunohistochemistry of Aβ and Morris water maze. Heavy burden of Aβ load and impaired Morris water maze performace were observed proving suitable for the experiment.Morris water maze of APP/PS1 and WT mice:After different interventions, Morris water maze was employed for behavioral tests. The whole process consisted of one day for adaption (no platform),5 consecutive days for acquisition training trials and one day for spatial probe trial.3. Preparation of Aβ1-42 oligomersThe preparation followed the instruction of Professor William J. Bowers (Rochester Medical Center). Aβ1-42 oligomers were identified by transmission electron microscopy and stored at -80℃. Serum-free medium was used to dilute Aβ1-42 oligomers into appropriate concentration for experimental usage.CCK-8 was used to detect cytotoxicity of different interventions for PC 12 cells.4. The detection of protein expression and phosphorylation statusWestern blotting was employed to detect changes of protein expression and phosphorylation status after different interventions for PC 12 cells, primary neurons, WT and APP/PS1 mice to determine the changes of ERS, JNK activation, insulin signaling status and Tau hyperphosphorylation. ①ERS markes:BiP, CHOP; ② JNK activation:t-JNK, p-JNK Thr183/Tyr185; ③ Insulin signaling status:t-1RS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, t-Akt, p-Akt Ser473;④ Pathological changes of AD:t-Tau and p-Tau Thr181. 5. The observation of mRNA levels for ERS geneReal-time quantitative PCR was employed to analyze changes in Aβ1-42 oligomers-treated PC12 cells. BiP, CHOP, ATF4, GADD34, Nrf2, PUMA, TRB3, XBP1s and XBP1u were regarded as ERS-related genes to evaluate the ERS activation.6. Statistical analysisQuantitative data were expressed as mean ± SD. t-test was used to compare difference between two groups. One-way ANOVA was performed for multiple comparisons using SPSS 17.0, followed by Tukey post hoc test. Statistical significance was established as ap value of less than 0.05.Results:1. AP1-42 oligomers impaired insulin signaling by increaseing IRS-1 serine phosphorylation. To evaluate the status of insulin signaling, basal insulin stimulation was needed. To detect the appropriate insulin concentration as the stimulation of the insulin signaling pathway, PC 12 cells were interposed with different concentrations (0,25,50,100 and 200 nM) of insulin for 30 min. The results showed that IRS-1 serine phosphorylations at 307,318 and 612 increased in a dose-dependent manner in PC 12 cells. After insulin stimulation (25 nM), phosphorylation of Akt on seine 473 significantly increased compared to that in the control group (p< 0.05), whereas the phosphorylation of serines 307,318 and 612 did not change significantly. So for the following experiment,25 nM insulin was used as basal insulin stimulation. As for the primary neurons,1 nM insulin was reported to be basal level.To explore the effect of Aβ1-42 oligomers on insulin signaling in PC12 cells, we employed various concentrations (0,0.25,0.5,0.75,1 and 2μM, for 120 min) and time gradients (0,15,30,60,120 and 240 min, at 1 μM) for Aβ1-42 oligomers treatment. Insulin stimulation (25 nM) was added 30 min before protein preparation. The Aβ1-42 oligomers treatment significantly increased the phosphorylation of IRS-1 on serines 307,318, and 612 in a dose-dependent and a time-correlated manner. The Aβ1-42 oligomers treatment (1 μM,120 min) significantly induced the serine phosphorylation on seines 307,318 and 612 (p< 0.05) and significantly disturbed the Akt seine 473 phosphorylation (p< 0.05) compared with that in the control group. The Aβ1-42 oligomers treatment (1 μM,120 min) was used for the following treatment for PC 12 cells. As for neurons, the Aβ1-42 oligomers treatment (500 nM,3 h) was refered to the references.2. AP1-42 oligomers and TG induced ERS, activated JNK, and impaired insulin signaling.In PC 12 cells, Aβ1-42(1 μM,120 min) treatment may cause elevated expression of BiP, CHOP with increased mRNA level of BiP, CHOP, ATF4, TRB3, PUMA, GADD34, Nrf-2 and XBP1 splicing (p< 0.05). Aβ1-42 (1 μM,120 min) and ERS inducer TG (2 μM,120 min) may cause increased phosphorylation of p-JNKThr183/Tyr185, p-IRS-1Ser307, p-IRS-1Ser318 and p-IRS-1Ser612(p< 0.05). Aβ1-42 (1 μM,24 h) and TG (2 μM,24 h) can cause elevated p-Tau Thr phosphorylation (p < 0.05).3. APP/PS1 mice exhibited ERS activation, JNK activation, and insulin signaling impairment compared to WT control.The cortex of 9-month-old male APP/PS1 mice showed increased BiP and CHOP expression (p< 0.05), elevated level of p-JNKThr183/Tyr185 (p< 0.05), multiple sites of serine phosphorylation at 307,318 and 612 (p< 0.05) compared with the same-month-old male WT littermates.4. ERS mediated the activation of JNK and insulin signaling impairment.In PC 12 cells and cortical primary neurons, pretreatment of ERS inhibitor PBA (2 mM,3 h) might partially reverse the Aβ1-42 oligomers induced p-JNKThr183/Tyr185, p-IRS-1Ser307, p-IRS-1Ser318, p-IRS-1Ser612 and p-TauThr181 phosphorylation (p< 0.05). APP/PS1 mice injected by intraperitoneal injection of PBA (200 mg/kg,5 weeks) significantly improved Morris water maze performance accompanied by reduced p-JNKThr183/Tyr185, p-IRS-1Ser307, p-IRS-1Ser318, p-IRS-1Ser612 phosphorylation (p< 0.05) in cortex.5. The underlying mechanism of ERS impairing insulin signaling might be JNK-dependent serine phosphorylation of IRS-1.In PC 12 cells and cortical primary neurons, pretreatment of JNK inhibitor SP600125 (25 μM,40 min) might partially alleviate the Aβ1-42 oligomers-induced p-JNKThr183/Tyr185, p-IRS-1Ser307, p-IRS-1Ser318, p-IRS-1Ser612 and p-TauThr181 phosphorylation. APP/PS1 mice injected by intraperitoneal injection of SP600125 (30 mg/kg,12 weeks) significantly improved Morris water maze performance accompanied by reduced p-JNKThr183/Tyr185, p-IRS-1Ser307, p-IRS-1Ser318, p-IRS-1Ser612 phosphorylation (p< 0.05) in cortex.Conclusions:1. ERS and insulin signaling impairment were observed in Aβ142 oligomers-treated PC 12 cells, cortical primary neurons and APP/PS1 mice.2. ERS accelerated insulin signaling impairment in Aβ1-42 oligomers-treated PC 12 cells, cortical primary neurons and and APP/PS1 mice.3. ERS accelerated insulin signaling impairment mediated by JNK-dependent IRS-1 serine phosphorylation in AP1-42 oligomers-treated PC 12 cells, cortical primary neurons and and APP/PS1 mice.4. ERS/JNK/IRS-1 might be involved in Tau hyperphosphorylation in Aβ1-42 oligomers-treated PC12 cells and cortical primary neurons. |
