Acute stress increases interstitial fluid amyloid-beta via corticotropin-releasing factor and neuronal activity

Abstract

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is critical in the pathogenesis of Alzheimer's disease. Abeta is produced by neurons and released into the brain interstitial fluid (ISF), a process regulated by synaptic activity. To determine whether behavioral stressors can regulate ISF Abeta levels, we assessed the effects of chronic and acute stress paradigms in amyloid precursor protein transgenic mice. Isolation stress over 3 months increased Abeta levels by 84%. Similarly, acute restraint stress increased Abeta levels over hours. Exogenous corticotropin-releasing factor (CRF) but not corticosterone mimicked the effects of acute restraint stress. Inhibition of endogenous CRF receptors or neuronal activity blocked the effects of acute stress on Abeta. Thus, behavioral stressors can rapidly increase ISF Abeta through neuronal activity in a CRF-dependent manner, and the results suggest a mechanism by which behavioral stress may affect Alzheimer's disease pathogenesis.

Fig. 1.

Effects of 3 months of isolation stress on soluble Aβ levels within the ISF, tissue lysates, and APP fragments in the hippocampus. (A) Three months of isolation stress increased ISF Aβ levels to 184 ± 23% of control levels in 4-month-old Tg2576 mice (P = 0.0006; n = 10 per group). In vivo concentrations of ISF Aβ in the hippocampus were 5,309 ± 145.0 and 2,881 ± 61.0 pg/ml in mice exposed to 3 months of isolation and control condition, respectively (data not shown). To assess the levels of soluble Aβ in the hippocampus, hippocampal tissues were processed at the end of 3 months of isolation and under control conditions. As determined by ELISA, both Aβ₄₀ (B) and Aβ₄₂ (C) were elevated by 37.9 ± 4.4% and 57.7 ± 9.4%, respectively in a carbonate-soluble fraction of hippocampal lysates from mice after 3 months of isolation stress vs. controls (P = 0.02; n = 7–8 per group). The same tissues were also assessed for the levels of full-length APP (FL-APP), APP α-CTF and APP β-CTF (n = 7–8 per group). (D) Representative lanes from Western blots for FL-APP, α-CTF, and β-CTF. The levels of FL-APP, α-CTF, and β-CTF were not changed after 3 months of isolation stress compared with control. Each band was normalized to the amount of α-tubulin in each lane. Data represent mean ± SEM.

Fig. 2.

Effects of 3 h of restraint stress on soluble Aβ levels within the ISF and tissue lysates, and APP fragments in the hippocampus. (A) Three hours of acute restraint stress increased ISF Aβ levels to 132 ± 6.9% of baseline by 13 h after the beginning of stress initiation in 3- to 4-month-old Tg2576 mice (P = 0.003; n = 10 per group). Hippocampal tissues were processed at 14 h after the beginning of 3 h of restraint stress initiation vs. the control condition. There were no significant differences in the levels of either Aβ₄₀ (B) or Aβ₄₂ (C) in stressed vs. control mice in the carbonate-soluble fraction of the tissue lysates as measured by ELISA (n = 8 per group). To determine whether APP processing was altered in stressed mice, the same tissues were also assessed for the protein expression levels of FL-APP, APP α-CTF, and APP β-CTF. (D) Representative lanes from Western blots for FL-APP, α-CTF, and β-CTF are shown. The levels of FL-APP and β-CTF were not different between groups. The levels of α-CTF were significantly decreased by 17.23 ± 3.404% in Tg2576 mice after 3 h of restraint stress compared with controls (P = 0.0005; n = 8 per group). Each band was normalized to the amount of α-tubulin in each lane. Data represent mean ± SEM.

Fig. 3.

Systemic administration with corticosterone (CORT) did not acutely alter ISF Aβ levels. The effect of a high dose of CORT on hippocampal ISF Aβ levels in 3- to 4-month-old Tg2576 mice is shown. After the basal ISF Aβ levels were obtained for 10 h, animals received an i.p. injection of 50 mg/kg CORT. An equal volume of vehicle solution (100 μl of 15% 2-hydroxypropyl-β-cyclodextrin in water) was used for control. There was no difference in ISF Aβ levels in CORT-treated vs. vehicle-treated mice (n = 8 per group).

Fig. 4.

Effects of CRF on ISF Aβ levels. To examine the effect of CRF on hippocampal ISF Aβ levels, 100 and 200 nM CRF were administrated by reverse microdialysis in the hippocampus of 3- to 4-month-old Tg2576 mice. (A) One hundred nanomolar CRF in the microdialysis fluid resulted in an increase ISF Aβ levels at 3 h after drug infusion, whereas 200 nM CRF increased ISF Aβ levels immediately after drug infusion (n = 5 per group). (B) Both 100 and 200 nM CRF increase ISF Aβ levels in a dose-dependent manner, reaching 138.3 ± 7.027% and 171.9 ± 17.83% of baseline by 12 h, respectively (P < 0.0001 and P = 0.0001, respectively). (C) Three-hour restraint stress increased ISF Aβ levels to 132 ± 6.896% compared with baseline by 13 h after the beginning of stress initiation (P = 0.003; n = 10 for stress). Treatment with α-helical CRF_9–41 (αCRF_9–41), a CRF receptor antagonist, given from 30 min before restraint stress until the end of the experiment, blocked the stress-induced increase in ISF Aβ levels (P = 0.006; n = 5 for stress + αCRF_9–41).

Fig. 5.

Neuronal/synaptic activity is involved in the stress-induced increase in ISF Aβ levels. Infusion with 5 μM TTX in the hippocampus by reverse microdialysis immediately decreased ISF Aβ levels, reaching 58.5% of baseline by 17 h from drug treatment in 3- to 4-month-old Tg2576 mice. Three hours of restraint stress was given to mice at 8 h after TTX treatment, which resulted in no significant change in ISF Aβ levels compared with controls treated with TTX alone controls (n = 5 per group).