Effects of corticotrophin-releasing factor receptor 1 antagonists on amyloid-β and behavior in Tg2576 mice

Abstract

Rationale: Previous studies indicate that psychosocial stressors could accelerate amyloid-β (Aβ) levels and accelerate plaque deposition in mouse models of Alzheimer disease (AD). Stressors enhanced the release of corticotrophin-releasing factor (CRF), and exogenous CRF administration mimicked the effects of stress on Aβ levels in mouse models of AD. However, whether CRF receptor 1 (CRF1) antagonists could influence the stress-induced acceleration of an AD-like process in mouse models has not been well studied.

Objective: We sought to examine whether CRF1 antagonists inhibit the effects of isolation stress on tissue Aβ levels, Aβ plaque deposition, and behaviors related to anxiety and memory in Tg2576 mice, and to investigate the molecular mechanism underlying such effects.

Methods: Cohorts of Tg2576 mouse pups were isolated or group-housed at 21 days of age, and then the subgroups of these cohorts received daily intraperitoneal injections of the CRF1 antagonists, antalarmin or R121919 (5, 10, and 20 mg/kg), or vehicle for 1 week. Other cohorts of Tg2576 mouse pups were isolated or group-housed at 21 days of age, and then at 4 months of age, subgroups of these mice were administered antalarmin (20 mg/kg) or vehicle in their drinking water for 6 months. Finally, cultured primary hippocampal neurons from regular Tg2576 pups (P0) were incubated with CRF (0.1, 1, and 10 nM), antalarmin (100 nM) or H-89 (1 μM) for 48 h. Brain tissues or cultured neurons were collected for histological and biochemical analyses, and behavioral measures were collected in the cohorts of mice that were chronically stressed.

Results: Administration of antalarmin at 20 mg/kg dose for 1 week significantly reduced Aβ1-42 levels in isolation stressed mice. Administration of antalarmin for 6 months significantly decreased plasma corticosterone levels, tissue Aβ1-42 levels, and Aβ plaque deposition in the brain and blocked the effects of isolation stress on behaviors related to anxiety and memory. Finally, incubation of neurons with 100 nM antalarmin inhibited the ability of 10 nM CRF to increase Aβ1-42 levels and protein kinase A IIβ expression. The effect of CRF1 on Aβ1-42 levels was also diminished by treatment with H-89, a c-AMP/PKA inhibitor.

Conclusions: These results suggest that CRF1 antagonists can slow an AD-like process in Tg2576 mice and that the c-AMP/PKA signaling pathway may be involved in this effect.

Figure 1. Antalarmin and R121919 reduce tissue Aβ levels in sub-acute stressed Tg2576 mice

Animals with one week of exposure to isolation stress (isolated) were associated with a significant increase in Aβ_1-40 (A, ##P<.001) and Aβ_1-42 levels (B: ## P< .001) as compared to group housed (Grouped) mice. Administration of antalarmin showed dose-dependent decreases of Aβ_1-42 levels in isolated mice. Post-hoc testing indicated that administration of antalarmin at a dose of 20mg/kg, but not at 10 mg/kg and 5mg/kg, significantly reduced levels of Aβ_1-42 (B: *P<.05, drug versus vehicle).

Figure 2. Antalarmin reduces tissue Aβ_1-42 levels and Aβ plaque deposition in chronically stressed Tg2576 mice

Tissue Aβ levels. Chronic isolation stress significantly increased Aβ_1-42 levels as compared to grouphoused animals in the cortex and hippocampus (A, B: #P< .05) at ten months of age. However, chronic administration of antalarmin (20 mg/kg) significantly decreased Aβ_1-42 levels as compared to the vehicle in isolated mice (A, B: *P< .05). Aβ plaque deposition. The number of Aβ immunohistochemical positive plaques in the cortex and hippocampus (C, D: ## P< .01, #P< .05) were significantly increased in isolated mice as compared to group-housed mice. Antalarmin administration (20 mg/kg) significantly decreased Aβ plaque deposition in the cortex of isolated mice (C: *P<.01: Isolated +Ant versus Isolated +Veh) and decreased Aβ plaque deposition in the hippocampus of both isolated mice; and group-housed mice (D: *P< .05, Isolated +Ant versus Isolated +Veh; ★P<.05: Grouped +Ant versus Grouped + Veh). Representative Aβ-immunocytochemical stained sections are shown from the cortex of 10-month-old Tg2576 mice from housed in isolation (Isolated, E, G) or under group-housed conditions (Grouped, F, H) and treated with antalarmin. Arrowheads indicate the plaques. Scale Bar =200 μm shown in panel H also applies to panels E, F, and G.

Figure 3. Antalarmin decreases plasma corticosterone levels in chronically stressed Tg2576 mice

Isolation housing beginning at weaning until 4 months of age significantly increased the plasma corticosterone levels at 4 months of age (#P<.001: Isolated+Veh vs. Grouped+Veh). Antalarmin administration significantly decreased the plasma corticosterone levels in isolated mice as compared to isolated vehicle group after 1 week (**P<.001) and after 6 months(* P< 0.05 Isolated + Veh vs. Isolated +Ant). Antalarmin had no significant effects on plasma corticosterone levels in group-housed mice.

Figure 4. Antalarmin blocks stress–induced increases in anxiety and memory-related behavior related behavior in Tg2576 mice

Chronic isolation stress significantly decreases the amount of time in the open field arm (A: #P<.01) and increases the number of entries in the closed arm compared to group-housed mice (B: #P<.05). Antalarmin administration ameliorated isolation stress–induced anxious behavior by increasing the duration in the open arm and decreasing the number of entries in the closed arm compared to isolated vehicle-treated mice (A, B: *P<.01). After chronic isolation stress, Tg2576 mice demonstrated less alternation and decreased rate of correct alternation as compared to group-housed mice (C, D: #P<.05). Again, antalarmin administration significantly restored number (C) and percentage (D) of correct alternation behaviors as compared to the isolated vehicle group (C, D: *P<.01). There was no difference in the total arm entries between isolated and group-housed animals.

Figure 5. Antalarmin inhibits the effect of CRF on Aβ_1-42 levels through the cAMP/PKA signaling pathway

Primary hippocampal neurons derived from Tg2576 mice cultured at 16 days without CRF (A) and with CRF for 48 hours (B). ELISA analysis indicates that 10nM but not 0.01nM or 1nM CRF administered to medium of primary neurons at 14 days significantly increased Aβ_1-42 levels after 48 hours of CRF exposure as compared to vehicle control (C: #P<.05). Treatment with 10nM CRF plus 100nM antalarmin blocked the increases of Aβ_1-42 levels compared to CRF treated groups (D: #P<.001: CRF vs. Veh; *P<.01: CRF vs CRF+Ant). H-89, a PKA inhibitor, co-treated with CRF blocked CRF effect on Aβ_1-42 levels (E: ★P<.05: CRF vs CRF+H-89). Western blotting analysis indicates that CRF significantly increased PKAIIβ expression, and antalarmin can block this effect. Treatment of PKA inhibitor H-89 also blocked CRF induced increase of PKAIIβ expression (F, G: #P<.001: CRF vs. Veh; *P<.05: CRF vs CRF+Ant; ★★P<.01: CRF vs CRF+H-89). Linear regression analysis indicates that Aβ_1-42 levels are highly correlated with PKAIIβ expression (J: r² = 0.72, P<.0001).