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. 2011 Jun 16;184:139-50.
doi: 10.1016/j.neuroscience.2011.03.067. Epub 2011 Apr 6.

Restraint Stress and Repeated Corticotrophin-Releasing Factor Receptor Activation in the Amygdala Both Increase Amyloid-β Precursor Protein and Amyloid-β Peptide but Have Divergent Effects on Brain-Derived Neurotrophic Factor and Pre-Synaptic Proteins in the Prefrontal Cortex of Rats

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Free PMC article

Restraint Stress and Repeated Corticotrophin-Releasing Factor Receptor Activation in the Amygdala Both Increase Amyloid-β Precursor Protein and Amyloid-β Peptide but Have Divergent Effects on Brain-Derived Neurotrophic Factor and Pre-Synaptic Proteins in the Prefrontal Cortex of Rats

B Ray et al. Neuroscience. .
Free PMC article

Abstract

Both environmental stress and anxiety may represent important risk factors for Alzheimer's disease (AD) pathogenesis. Previous studies demonstrate that restraint stress is associated with increased amyloid beta (Aβ) and decreased brain-derived neurotrophic factor (BDNF) levels in the brain. Aβ deposition, synaptic loss, and neurodegeneration define major hallmarks of AD, and BDNF is responsible for the maintenance of neurons. In contrast to restraint stress, repeated injections of sub-anxiogenic doses of the corticotrophin releasing factor receptor agonist urocortin1 (Ucn1) administered in the basolateral amygdala (BLA) of rats elicits persistent anxiety-like responses. We hypothesized that both restraint stress and Ucn1-induced anxiety would contribute to a neurobiological abnormality that would change the levels of Aβ precursor protein (APP) and Aβ as well as BDNF and pre-synaptic markers. In the first experiment, adult male Wister rats (n=5) were subjected to 3-h restraint, as compared to unstressed controls. In the second experiment, adult male Wistar rats (n=6) were subjected to sub-anxiogenic doses of Ucn1 (6 fmol/100 nl) administered in the BLA for 5 consecutive days, as compared to controls. Following each respective treatment, the social interaction (SI) test was performed to measure anxiety-like behavior. Protein studies were then conducted to quantify levels of APP, Aβ, BDNF and presynaptic proteins in the prefrontal cortex (PFC). In both experiments, we detected differences in either corticosterone levels or the SI test associated with a stress response. Furthermore, our findings indicate that both restraint stress and Ucn1 administration in the BLA lead to increased APP and Aβ deposition. However, restraint-induced stress leads to reductions in the levels of BDNF and presynaptic markers, while Ucn1-induced anxiety is associated with increases in the levels of each respective protein. This demonstrates a convergent role for stress response and Ucn1-induced anxiety in the regulation of APP and Aβ, but opposing roles for each respective treatment in the regulation of BDNF and presynaptic markers.

Figures

Figure 1A
Figure 1A. Social interaction of control and stressed animals after three hrs of restraint stress
The social interaction test was conducted to measure anxiety-like behavior in rats. Following three hrs of restraint stress, we have observed a trend in decreased social interaction in stressed rats (P=0.06 by one tailed t-test). Figure 1B: Social interaction of vehicle and Ucn1-treated animals: Following repeated Ucn1 injections into the BLA, the rats showed significant decreases in social interactions consistent with increased anxiety-like behavior. Figure 1C: Plasma corticosterone levels in control and restraint stressed animals: Plasma corticosterone was measured after three hrs restraint stress from both stressed and control animals. A competitive EIA assay was utilized to measure corticosterone and the levels were significantly higher in the stressed animals versus controls.
Figure 1A
Figure 1A. Social interaction of control and stressed animals after three hrs of restraint stress
The social interaction test was conducted to measure anxiety-like behavior in rats. Following three hrs of restraint stress, we have observed a trend in decreased social interaction in stressed rats (P=0.06 by one tailed t-test). Figure 1B: Social interaction of vehicle and Ucn1-treated animals: Following repeated Ucn1 injections into the BLA, the rats showed significant decreases in social interactions consistent with increased anxiety-like behavior. Figure 1C: Plasma corticosterone levels in control and restraint stressed animals: Plasma corticosterone was measured after three hrs restraint stress from both stressed and control animals. A competitive EIA assay was utilized to measure corticosterone and the levels were significantly higher in the stressed animals versus controls.
Figure 2A
Figure 2A. Levels of total APP in the frontal cortex of control and restraint stressed animals
Frontal cortex of control and stressed animals were homogenized in Tris- HCl buffer supplemented with protease inhibitors cocktail. Equal amount of protein samples from both control and stressed groups were loaded in 10% polyacrylamide mini gel, and Western immunoblot was performed to measure the levels of total APP. APP band density was normalized with β-actin bands. Normalized APP bands showed a significant increase in the frontal cortex of restrained stressed animals versus controls. Figure 2B: Levels of total APP in the frontal cortex of control and Ucn1-treated animals: Western immunoblot analysis of frontal cortex brain lysate from vehicle and Ucn1-treated animals was performed as described previously in ‘Figure 3A’. Normalized APP band density showed a significant increase in frontal cortex of Ucn1-treated animals versus controls.
Figure 2A
Figure 2A. Levels of total APP in the frontal cortex of control and restraint stressed animals
Frontal cortex of control and stressed animals were homogenized in Tris- HCl buffer supplemented with protease inhibitors cocktail. Equal amount of protein samples from both control and stressed groups were loaded in 10% polyacrylamide mini gel, and Western immunoblot was performed to measure the levels of total APP. APP band density was normalized with β-actin bands. Normalized APP bands showed a significant increase in the frontal cortex of restrained stressed animals versus controls. Figure 2B: Levels of total APP in the frontal cortex of control and Ucn1-treated animals: Western immunoblot analysis of frontal cortex brain lysate from vehicle and Ucn1-treated animals was performed as described previously in ‘Figure 3A’. Normalized APP band density showed a significant increase in frontal cortex of Ucn1-treated animals versus controls.
Figure 3A & 3B
Figure 3A & 3B. Levels of Aβ (x-40) in the cortical lysates
To determine the levels of Aβ (x-40) in the cortical lysates, a sensitive chemiluminescent ELISA was used. This sandwich ELISA measures Aβ (x-40) in rodent and human brain samples and has negligible cross reactivity with Aβ (x-42). The ELISA values were normalized by the total protein content of the lysates and plotted as ‘% control’. Normalized results revealed a significant increase in the levels of Aβ (x-40) in the cortical lysates of both restraint stressed and Ucn1-treated animals versus control/vehicle treated animals. Figure 3C and 3D: Levels of Aβ (x-42) in the cortical lysates: Aβ (x-42) levels in the cortical lysates were measured by a sensitive and specific chemiluminescent ELISA as per manufacturer's protocol. Aβ (x-42) signals were normalized and plotted in the same way as described in ‘Figure 3A and 3B’. Normalized Aβ (1-42) showed a significant increase in the cortex of restraint stressed animals versus controls. However, Aβ (x-42) levels did not differ between the cortical lysates of vehicle and Ucn1-treated animals.
Figure 3A & 3B
Figure 3A & 3B. Levels of Aβ (x-40) in the cortical lysates
To determine the levels of Aβ (x-40) in the cortical lysates, a sensitive chemiluminescent ELISA was used. This sandwich ELISA measures Aβ (x-40) in rodent and human brain samples and has negligible cross reactivity with Aβ (x-42). The ELISA values were normalized by the total protein content of the lysates and plotted as ‘% control’. Normalized results revealed a significant increase in the levels of Aβ (x-40) in the cortical lysates of both restraint stressed and Ucn1-treated animals versus control/vehicle treated animals. Figure 3C and 3D: Levels of Aβ (x-42) in the cortical lysates: Aβ (x-42) levels in the cortical lysates were measured by a sensitive and specific chemiluminescent ELISA as per manufacturer's protocol. Aβ (x-42) signals were normalized and plotted in the same way as described in ‘Figure 3A and 3B’. Normalized Aβ (1-42) showed a significant increase in the cortex of restraint stressed animals versus controls. However, Aβ (x-42) levels did not differ between the cortical lysates of vehicle and Ucn1-treated animals.
Figure 4A
Figure 4A. Levels of BDNF in the frontal cortex of control and restraint stressed animals: Western immunoblot analysis
Acid treated frontal cortex lysates were analyzed by western immunoblotting to detect the levels of BDNF (for details see ‘Materials and Methods’). BDNF band densities (primarily BDNF dimer) were normalized by β-actin signals. Normalized BDNF showed a significant decrease in the cortex of animals underwent 3 hrs restraint stress versus controls. Figure 4A: Levels of BDNF in the frontal cortex of control and restraint stressed animals: ELISA analysis. To confirm the BDNF Western immunoblot results, BDNF levels were measured in acid treated cortical lysates by a sensitive colorimetric ELISA. BDNF values (pg/ml) were converted to pg/μg, normalized by protein contents of the lysates and plotted. BDNF ELISA also revealed a significant decrease in the cortex of restraint stressed animals versus controls.
Figure 5A
Figure 5A. Levels of BDNF in the frontal cortex of vehicle and Ucn1-treated animals: Western immunoblot analysis
BDNF Westernblotting was performed with the cortical lysates of vehicle and Ucn1-treated animals in a similar way described in ‘Figure 4A’. β-actin adjusted BDNF band densities (BDNF dimer) showed a significant increase in the cortical lysates of Ucn1-treated animals versus vehicle. The lower band observed in this figure is due to secondary ‘artifact’ and excluded from the analyses. Figure 5B: Levels of BDNF in the frontal cortex of vehicle and Ucn1-treated animals: ELISA analysis. To confirm the findings of ‘Figure 3A’, a sensitive ELISA was used to determine the levels of BDNF in the acid treated cortical lysates of vehicle and Ucn1-treated animals. BDNF values (pg/ml) were converted to pg/μg, normalized by protein contents of the lysates and plotted. Results showed a significant increase in the cortical lysates of Ucn1-treated animals versus vehicle.
Figure 6A
Figure 6A. Levels of syntaxin6 in the cortical lysates of control and restraint stressed animals
Western immunoblot analyses of cortical lysates of control and restrained stressed animals were carried out with specific monoclonal syntaxin6 antibody, which recognizes one of the important pre-synaptic proteins syntaxin6. The bands were normalized by β-actin signals. Normalized syntaxin6 bands showed a significant decrease in the lysates of restraint stressed animals versus controls. Figure 6B: Levels of SNAP25 in the cortical lysates of control and restraint stressed animals: Western immunoblot analyses of the same samples mentioned in ‘Figure 6A’ for another pre-synaptic protein SNAP25 showed a decreasing trend (p=0.08) in the cortical lysates of restraint stressed animals versus controls.
Figure 7A
Figure 7A. Levels of SNAP25 in the cortical lysates of vehicle and Ucn1-treated animals
Western immunoblot analyses of cortical lysates of vehicle and Ucn1-treated animals were carried out to detect the levels of cortical SNAP25. SNAP25 bands were normalized by β-actin signals and plotted. Normalized SNAP25 showed a significant increase in the cortex of Ucn1-treated animals versus vehicle treated animals. Figure 7B: Levels of syntaxin6 in the cortical lysates of vehicle and Ucn1-treated animals: Western immunoblot analyses revealed a significant increase in the levels of syntaxin6 in the cortical lysates of Ucn1-treated animals versus vehicle treated animals.

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