The Effect Of Chronic Stress On The Cognitive Function And Metabolic Profiling In APP/PS1 Double Transgenic Mice

Stress or stress response is a nonspecific response of the body to various stimuli, including endogenous or exogenous stressors. Moderate stress is beneficial to face some sudden dangers and strengthen the adaptive capacity of body, while drastic or chronic stress can lead to excessive reaction of psychological or/and physiological, finally affect the quality of life and health.Except the strength and the duration, stress response is also associated with the physical condition, individual experience and cognitive evaluation, as well as the support of family and society. Brain is the central orange of stress response.When subjected to stress, the healthy brain regulates the nervous, endocrine and immune systems to maintain allostasis; however, in many conditions, such as aging, illness or genetic mutations, that result in brain dysfunction(referred to hereafter as “fragile brain”), even low stress intensity can lead to allostatic overload. In addition, a growing number of experiments shown that the brain is also one of the important target organs of stress response. Chronic stress can decreased the nerve branches, dendritic spines and synaptic number in hippocampus, amygdala and prefrontal cortex, but also impaired the cognitive function, affected the regulation of emotion and autonomous behavior, these damages were more serious in the elderly or diseased. However, it is still not very clear that the effect of chornic stress to the brain structure and function in individuals with different genotype.The hypothalamic–pituitary–adrenal(HPA) axis is a major neuroendocrine stress axis whose activation culminates in increased levels of glucocorticoids(GCs; primarily cortisol in humans, and corticosterone in rodents). The hippocampus is a key structure that not only is involved in learning and memory but is also regulated by the negative feedback of the HPA axis, as it contains some of the highest GR levels in the brain. With thecombination of the hormones and antibodies, hippocampus can feel the glucocorticoid levels in the body and adjust the activity of HPA through the negative feedback mechanism, and it is one of the most vulnerable organization during overload stress(e.g., chronic stress). This negative feedback of HPA axis was suppressed when the level of glucocorticoid receptors was reduced in the brain of aging or disease states. At this point, the active HPA axis caused by stress was difficult to restore to the basic level and produced excessive amounts of glucocorticoid. Excessive glucocorticoid are known to exert noxious effects on the central nervous system, especially the hippocampus, including causing dendritic atrophy, inhibiting neurogenesis,and processes that ultimately contribute to cognitive impairment and/or development of Alzheimer’s disease(AD). Many studies highlight the damage effect of abnormal glucocorticoid secretion for brain may related to insulin and insulin-associated signaling pathway. Moreover, the brain will produce a series of comprehensive response under the interaction between hormones and multiple signaling pathways, resulting in changing the metabolic patterns.In this study, we used APP/PS1 double transgenic and wild-type mice, via chronic unpredictable mild stress(CUMS) model to simulate the stress response to a variety of environmental and psychological stressor. The aim of this study is to determine whether the presence of AD-associated genetic mutations affect stress tolerance, cognitive function, biochemical progression and the cognitive-related metabolic profiling.Part one The effect of chronic stress on the cognition and the ultrastructure of neuron in APP/PS1 double transgenic miceObjective: To observe the effect of chronic stress on the cognition and the ultrastructure of neuron in wild-type and APP/PS1 double transgenic mice,and explore the genetic influences on the stress tolerance of brain.Methods:1 Male 6-month-old APPswe/PSEN1 d E9(for short, APP/PS1) and C57 mice in four groups(C57, C57+CUMS, APP/PS1 and APP/PS1+CUMS groups) were used. CUMS mice were exposed to various randomly scheduled,low-intensity social and environmental stressors 2-3 times a day for 4 weeks.2 Weigh the mice at before, middle and after of the CUMS experiment to reflect the general condition.3 The cognitive behavioral tests(NOR and MWM) were performed in the sequence as follows, beginning on the day following the 3 weeks of CUMS.4 After CUMS, congo red stain was used to determine the amyloid plaques, and ELISA were used to detect the expression level of Aβ40 and Aβ42 in hippocampus.5 TEM was applied to observe the neurons and synaptic morphology in hippocampus CA1 area of different group mice, and analysis the parameter of synapsis.Results:1 Before the start of the experiment, there was no difference no the weight among the mice of four groups(P>0.05). At the middle of the experiment, compared with the C57 mice, the weight of C57+CUMS mice and APP/PS1 mice had no obvious difference(P>0.05); compared with the APP/PS1 mice, the weight of APP/PS1+CUMS mice was decreased(P=0.007). At the end of the experiment, compared with the C57 mice, the weight of C57+CUMS mice was significantly reduced(P<0.001); compared with the APP/PS1 mice, the weight of APP/PS1+CUMS mice was further decreased(P<0.001), n=13/group.2 During the training phase of NOR, there was no difference in time spent exploring two identical objects in both genotypes of mice(P>0.05).One-way ANOVA indicated the cognitive impairment in APP/PS1+CUMS mice(P=0.015), as the discrimination index was lower than that of APP/PS1 mice, while no defferences were found between other groups(P>0.05),n=13/group.3 The swim speed of MWM was not affected by genotype or CUMS(P>0.05). In the place navigation test, post-hoc tests showed that the escape latency was increased in APP/PS1 mice compared with C57 mice on the fourth(P=0.019) and fifth days(P=0.024); the escape latency was extended inAPP/PS1+CUMS compared to APP/PS1 mice on the third(P=0.008) and fifth days(P=0.001); and there were no differences between C57 and C57+CUMS mice(P>0.05). In the probe trial, post-hoc tests showed that, when compared to C57 mice, time spent in the target quadrant was significantly decreased in C57+CUMS mice(P=0.017) and APP/PS1 mice(P=0.001) and was further decreased in APP/PS1+CUMS mice(vs APP/PS1 mice P=0.007), n=13/group.4 As demonstrated by Congo red stain, there was no senile plaque deposition in C57 and C57+CUMS mice(P>0.05); the deposition was appeared in 6-month APP/PS1 mice compared with C57 mice(P=0.048); and it was aggregated in APP/PS1+CUSM mice(P=0.001), n=3/group. Consistent with this, the Aβ40 and Aβ42 ELISA results showed that the levels of Aβ40and Aβ42 were increased in 6-month-old APP/PS1 mice compared to C57mice(P= 0.035, P=0.002) and that CUMS further upregulated the levels of Aβ42 when comparing APP/PS1+CUMS and APP/PS1 mice(P=0.049), but no effect of CUMS on Aβ40 and Aβ42 levels were detected between wild-type mice(C57 vs C57+CUMS, P>0.05), n=8/group.5 TEM was used to observe the ultrastructure of hippocampal neurons. In C57 and C57+CUMS mice, the neuronal ultrastructure was normal as exhibited by intact membranes, uniform cytoplasm and complete organelle structure. In APP/PS1 mice, although there were no overt changes in organelle structure, a few lipofuscin deposits were observed. In APP+CUMS mice,neurons exhibited very poor cellular health, presenting chromatin margination,cytoplasm vacuolization, decreased cytoplasmic organelles, serious fusion of mitochondrial cristae and membrane, distention of the rough endoplasmic reticulum, and increased lipofuscin deposits and autophagosomes. The parameter of synapsis shown there was no different on the synaptic cleft(P>0.05), the synaptic curvature was significant decreased in C57+CUMS group compared with C57 group. Both C57+CUMS and APP/PS1 mice were statistically different from C57 mice(P<0.001, P<0.001), and the length of the PSD was further decrease in APP/PS1+CUMS group(vs APP/PS1 mice P=0.033), n=29-32/group.Part two The effect of chronic stress on insulin and insulin-relatedsignaling pathway in APP/PS1 double transgenic miceObjective: To discuss the change of insulin and insulin-associated signaling pathway in wild-type and APP/PS1 mice during chronic stress,understanding the lower stress tolerance in APP/PS1 mice and the molecular biology mechanism which is hidden in the brain.Methods:1 The model and group were similar to part one.2 After behavioral studies, animals underwent fasting overnight, and blood glucose levels were measured by a Roche glucose meter. Mice were then sacrificed and the serum was collected, corticosterone and insulin were detected using a commercially available Iodine [125I] Radioimmunoassay Kit.3 After behavioral studies, the brain was removed and embedded in paraffin, and then 5-μm-thick consecutive coronal paraffin sections.Immunohistochemistry was used to detect the expression of IRS1 and IRS1-p Ser636 in in hippocampus CA1 area.4 MILLIPLEX phospho-MAP mates for total β-tubulin, phosphor JNK/SAPK1-p Thr183/Tyr185, ERK/MAPK1/2-p Thr185/Tyr187. And MILLIPLEX MAP Akt/m TOR Total Phosphoprotein Magnetic Bead Kit was detect for the levels of Akt-p Ser473, GSK3α-p Ser21, GSK3β-p Ser9,IR-p Tyr1162/1163, IRS1-p Ser636, m TOR-p Ser2448, p70S6K-p Thr412,PTEN-p Ser380, TSC2-p Ser939 and RPS6-p Ser235/236.5 Western boltting analyzed the GR expression in hippocampus of mice,and further verified the alteration of Akt/m TOR signaling pathways in four groups.Results:1 The corticosterone levels were significantly increased in APP/PS1+CUMS mice compared with the APP/PS1 mice(P<0.001), but not different between C57 and C57+CUMS mice(P>0.05), n=8/group. The result of western boltting shown that the GR was significantly decreased in APP/PS1 mice compared with C57 mice(P<0.001), and CUMS could further decreasethe expression of GR(P=0.001), while no obvious different was found between C57 and C57+CUSM group(P>0.05), n=4/group.2 HOMA value was calculated using the fasting blood glucose and serum insulin, we found that it was obviously higher in APP/PS1+CUMS group than that in APP/PS1 group(P=0.018), and there was no difference in C57,C57+CUMS and APP/PS1 groups(P>0.05), n=8/group. Immunoassays &MILLIPLEX® MAP Kits(abbreviated here as “immunoassays”) showed he level of IR-p Tyr1162/1163 was significant decrease in APP/PS1 mice(vs C57 mice, P<0.001), and a further decrease in APP/PS1+CUMS mice(vs APP/PS1 mice, P=0.001). Total IRS1 protein was not different across the four groups(P>0.05). The levels of IRS1-p Ser636 were significant increase in APP/PS1 mice compared to the C57 group(IHC, P<0.001; and Immunoassay, P=0.001),and a further increase in APP/PS1+CUMS mice(vs APP/PS1 mice; IHC,P<0.001; and Immunoassay, P<0.001), n=7-8/group.3 We also found that, compared with APP/PS1 mice, the expression of JNK-p p Thr183/Tyr185 was increased in APP/PS1+CUMS mice(P=0.036).For AKT-p Ser473, m TOR-p Ser2448 and p70S6K-p Thr389/412 molecules, the result shown these molecules were decreased in the APP/PS1group(vs C57 group, P=0.002, P<0.001 and P=0.001,), and further decreased in the APP/PS1+CUMS group(vs APP/PS1 group, P=0.014, P=0.006 and P=0.008), but no difference was found in C57 and C57+CUMS group(P >0.05). And statistical analysis indicated the ERK-p Thr185/Tyr187 level were lower in APP/PS1 than C57 mice(P=0.044). We also examined levels of PTEN-p Ser380 and found that it was increased in the APP/PS1 group(vs C57 group, P<0.001). The expression of other molecules, for example GSK3α-p Ser21, GSK3β-p Ser9, TSC2-p Ser380 and RPS6-p Ser235/236 were no difference in the four groups(P>0.05), n=7-8/group.Western blotting confirmed that there were no difference on the expression of Akt-p Ser473 and m TOR-p Ser2448 between wild-type and APP/PS1 mice(P>0.05). Compared with the C57 mice, they were all decreased in APP/PS1 mice(P=0.002, P=0.018), and an enhancement effecton Akt-p Ser473 in APP/PS1+CUMS mice(P=0.002), n=4/group.Part three The metabolic profiling in APP/PS1 double transgenic miceduring chronic stressObjective: GC-TOF/MS technique was used to investigate whether the presence of AD-associated genetic mutations affect cognitive-related metabolic profiling during chronic stress.Methods:1 The model and group were similar to part one.2 After behavioral studies, the hippocampus were dissected on ice and stored at-80 °C. Weighting 10 mg hippocampal tissue for extraction and derivation, the mixture sample was subjected to detection by GC-TOF/MS,n=6/group.3 After data preprocessing, the resulted three-dimensional data involving the peak number, sample name, and normalized peak area were fed to SIMCA13.0 software package(Umetrics, Umea, Sweden) for principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA) and orthogonal projections to latent structures-discriminate analysis(OPLS-DA), and the remaining variables were then assessed by Student’s T test, P<0.05 was considered as statistically significant between two comparison groups.4 To find the cognitive-related metabolites during stress process, we assessed the correlation between the performance of MWM and the levels of metabolite, and the results of this analysis was selected by the |Pearson correlation|>0.5 and P<0.05.Results:1 Unsupervised principal component analysis showed a clear separation between every two groups, except C57 and C57+CUMS group. As a supervised clustering analysis, PLS-DA model was applied to understand the variables for classification. The R2 Y value of C57 vs C57+CUMS, C57 vs APP/PS1 and APP/PS1 vs APP/PS1+CUMS in PLS-DA analysis were 0.993,0.998 and 0.993, and the Q2 Y value were 0.574, 0.692 and 0.68, whichsuggested that the model were stable and good to fitness and prediction.Furthermore, 7-fold cross validation and 200 permutation tests were used to estimate the robustness and the predictive ability of our mode, the low values of Q2 intercept further validate the robustness of the models, and thus show a low risk of over fitting and reliable. To refine the PLS-DA analysis,OPLS-DA analysis was performed to maximize the differences between groups. In the score plots of OPLS-DA, all samples were inside the 95%Hotelling T2 ellipse and the samples from different groups were scattered into different regions. The VIP >1.0 were first selected, and if the similarity of the remaining variables > 700 indicates that the accuracy of the metabolite identification is reliable, finally, 24 metabolites were changed in C57 vs C57+CUMS, 25 metabolites were altered in C57 vs APP/PS1, and 33 metabolites were changed in APP/PS1 vs APP/PS1+CUMS.2 SPSS 13.0 was used to further analysis, under the non-stress condition,significant differences were found in the levels of 8 metabolites between C57 and APP/PS1 mice. On the one hand, the levels of valine, beta-alanine,o-phosphorylethanolamine and pantothenic acid were significantly decreased;on the other hand, the levels of sorbose, palmitic acid and stearic acid were increased in APP/PS1 mice. When suffered from CUMS, compared to C57 mice, 1-monopalmitin level was decreased, palmitic acid and stearic acid levels were increased in C57+CUMS mice. Compared to APP/PS1 mice, the levels of serine, threonine, o-phosphorylethanolamine and monostearin decreased significantly, in contrast, the levels of 3-hydroxybutyric acid,L-malic acid and pantothenic acid increased in APP/PS1+CUMS mice(P<0.05).3 The correlation between the performance of MWM and the levels of metabolites, and the results of this analysis was selected by the |Pearson correlation|>0.5 and P<0.05. We found 3-hydroxybutyric acid, valine, serine,beta-Alanine and O-Phosphorylethanolamine were the most closely with the cognitive-related metabolites during stress process.Conclusions:1 The effect of CUMS were different in APP/PS1 and wild-type mice on the aspect of cognition and neuropathology, implying 6-month C57 mice exhibited almost normal regulation to chronic stress, while age-matched APP/PS1 mice showed lower stress toleration. These results suggested that4-week CUMS had no obvious effect on the cognition and neuronal ultrastructure in C57 mice, while aggravated cognitive impairement, damaged neuronal ultrastructure, increased senile plaques and Aβ level in APP/PS1 mice.2 Compared with C57 mice, the corticosterone level was dramatically increased, and the GR expression and stress toleration were decreased in6-month APP/PS1 mice after 4-week CUMS, which is associated with insulin resistance and the reinforced inhibition of the Akt/m TOR signaling pathway mediated by insulin resistance in hippocampus. Therefore, stress management or hormone homeostasis is very important in these vulnerable groups.3 CUMS lead to metabolic disturbance in the brain of APP/PS1. Among these metabolites, 3-hydroxybutyric acid, valine, serine, beta-alanine and o-phosphorylethanolamine were closely associated with the cognitive impairment during CUMS process. These metabolites mainly involved in sphingolipid metabolism, synthesis and degradation of ketone bodies, and amino acid metabolism pathway.