Mitochondrial protection by the mixed muscarinic/σ1 ligand ANAVEX2-73, a tetrahydrofuran derivative, in Aβ25-35 peptide-injected mice, a nontransgenic Alzheimer's disease model

Abstract

Alzheimer's disease (AD), the most prevalent dementia in the elderly, is characterized by progressive synaptic and neuronal loss. Mitochondrial dysfunctions have been consistently reported as an early event in AD and appear before Aβ deposition and memory decline. In order to define a new neuroprotectant strategy in AD targeting mitochondrial alterations, we develop tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (ANAVEX2-73, AE37), a mixed muscarinic receptor ligand and a sigma-1 receptor (σ1R) agonist. We previously reported that ANAVEX2-73 shows anti-amnesic and neuroprotective activities in mice injected intracerebroventricular (ICV) with oligomeric amyloid-β25-35 peptide (Aβ25-35). The σ1R is present at mitochondria-associated endoplasmic reticulum (ER) membranes, where it acts as a sensor/modulator of ER stress responses and local Ca(2+) exchanges with the mitochondria. We therefore evaluated the effect of ANAVEX2-73 and PRE-084, a reference σ1R agonist, on preservation of mitochondrial integrity in Aβ25-35-injected mice. In isolated mitochondria from hippocampus preparations of Aβ25-35 injected animals, we measured respiration rates, complex activities, lipid peroxidation, Bax/Bcl-2 ratios and cytochrome c release into the cytosol. Five days after Aβ25-35 injection, mitochondrial respiration in mouse hippocampus was altered. ANAVEX2-73 (0.01-1 mg/kg IP) restored normal respiration and PRE-084 (0.5-1 mg/kg IP) increased respiration rates. Both compounds prevented Aβ25-35-induced increases in lipid peroxidation levels, Bax/Bcl-2 ratio and cytochrome c release into the cytosol, all indicators of increased toxicity. ANAVEX2-73 and PRE-084 efficiently prevented the mitochondrial respiratory dysfunction and resulting oxidative stress and apoptosis. The σ1R, targeted selectively or non-selectively, therefore appears as a valuable target for protection against mitochondrial damages in AD.

Keywords: ANAVEX2-73; Alzheimer’s disease; cytoprotection; mitochondrial damages; sigma-1 receptor.

Figure 1

ANAVEX2-73 and PRE-084 prevented oxidative stress in Aβ_25–35-treated mice. Mice were administered ICV with Sc. Aβ or Aβ_25–35 peptide (9 nmol) and sacrificed after 1, 3, 5 or 7 days for lipid peroxidation measures (A). n = 6 per group, ^*p < 0.05, ^***p < 0.001 vs. the (Sc. Aβ + V)-treated group at the same timepoint; t-test. Mice were then administered IP with (B) ANAVEX2-73 (0.01–1 mg/kg) or (C) PRE-084 (0.5–1 mg/kg) 20 min before Aβ_25–35 (9 nmol). In two groups, BD1047 (10 mg/kg) was administered simultaneously with the highest dose of each agonist. Mice were sacrificed after 7 days for lipid peroxidation measures. One-way ANOVA: F_(7,70) = 22.5, p < 0.0001, n = 6–14 per group in (B); F_(4,69) = 31.9, p < 0.0001, n = 6–22 in (C). ^**p < 0.01, ^***p < 0.001 vs. the (Sc. Aβ+V)-treated group; ^###p < 0.001 vs. the (Aβ_25–35+V)-treated group; Dunnett’s test.

Figure 2

ANAVEX2-73 (AN2-73) and PRE-084 (PRE) protected against Aβ_25–35-induced alteration of mitochondrial respiration in mice. Mice were treated with ANAVEX2-73 (0.3 mg/kg IP), PRE-084 (0.5 mg/kg IP) or vehicle, before injection of Sc. Aβ or Aβ_25–35 (9 nmol ICV). (A–C) Mitochondria (0.8 mg/ml) were loaded in the chamber with appropriate buffer at 30°C. State 2 respiration was activated by addition of pyruvate-malate (5 mM). State 3 respiration was induced with ADP. State 4 respiration was provoked by the addition of carboxyatractyloside (CAT), a blocker of ATP-ADP carrier. Finally, addition of tyrphostine, an uncoupling agent, activated the uncoupled respiration. The respiratory control ratio is the state 3/state 4 ratio. n = 4–8 per group, F_(5,38) = 7.11, p < 0.0001 for state 2 and F_(5,38) = 8.43, p < 0.0001 for state 4 in (A); F_(5,38) = 13.1, p < 0.0001 for state 3 and F_(5,38)= 8.43, p < 0.0001 for state 3 in (B); F_(5,38) = 2.58, p < 0.05 in (C). ^*p < 0.05, ^**p < 0.01 vs. Sc. Aβ/Veh; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Aβ_25–35/Veh. Dunnett’s test. (D) Typical trace of O₂ consumption for Sc. Aβ and Aβ_25–35-injected mice.

Figure 3

Direct application of ANAVEX2-73 or PRE-084 affected mitochondrial respiration only at high concentrations. Mitochondria (0.8 mg/ml) were loaded in the chamber with appropriate buffer at 30°C. The drugs (10⁻⁸ to 10⁻⁴ M) were bath applied 5 min before the repspiration measures. State 2 respiration was activated by addition of pyruvate-malate (5 mM). State 3 respiration was induced with ADP. State 4 respiration was provoked by the addition of carboxyatractyloside (CAT), a blocker of ATP-ADP carrier. The respiratory control ratio is the state 3/state 4 ratio. n = 4–8 per group (n = 14 for the no-drug group), F_(5,38) = 3.08, p < 0.05 for ANAVEX2-73 and F_(5,31) = 5.19, p < 0.01 for PRE-084 in (A); F_(5,38) = 22.1, p < 0.0001 for ANAVEX2-73 and F_(5,31)= 7.53, p < 0.001 for PRE-084 in (B). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. no-drug data; Dunnett’s test.

Figure 4

ANAVEX2-73 protected against Aβ_25–35-induced alteration of complex IV activity. Mitochondria were loaded in the spectrophotometer cuvette with appropriate buffer at 30°C and variations of absorption were recorded after addition of specific substrates. (A) Effect of Aβ_25–35 ICV injection on complex activities (% of Sc. Aβ) n = 8–9 per group, * p < 0.05 vs. Sc. Aβ Student’s t-test. (B) Mice were treated with ANAVEX2-73 (0.3 mg/kg IP) or vehicle, before injection of Sc. Aβ or Aβ_25–35 (9 nmol ICV). Complex IV activity was measured after 7 days (% of Sc. Aβ). n = 5–9 per group, F_(3,26) = 3.00, p < 0.05. * p < 0.05 vs. Sc. Aβ; Dunnett’s test.

Figure 5

ANAVEX2-73 and PRE-084 prevented Aβ_25–35-induced cytochrome c release. (A) Typical blots of mitochondrial (M) and cytosolic (C) fractions with cytochrome c, oxphos and β-tubulin antibodies. (B) Cytochrome c content (cytosol to mitochondrial content ratios). Mice were injected with Sc. Aβ or Aβ_25–35 (9 nmol ICV) and sacrificed at indicated days. n = 9–12 per group. * p < 0.05 vs. Sc. Aβ. Student’s t-test. (C, D) Mice were treated with ANAVEX2-73 (0.3 mg/kg IP) or PRE-084 (0.5 mg/kg IP) before Aβ_25–35 and sacrificed after 5 days. n = 8–17, F_(2,37) = 5.96, p < 0.01 in (C); n = 8–14, F_(2,33) = 8.19, p < 0.01 in (D). ^*p < 0.05, ^**p < 0.01 vs. Sc. Aβ, ^#p < 0.05, ^##p < 0.01 vs. Aβ_25–35; Dunnett’s test.

Figure 6

ANAVEX2-73 protected against Aβ_25–35-induced increase in Bax/Bcl-2 ratio in the mouse hippocampus. (A–C) Mice were sacrificed at 1, 3, 5 and 7 days after Aβ_25–35 injection and Bax and Bcl-2 contents determined by western blots. (D–F) Effect of ANAVEX2-73 (0.1–1 mg/kg IP) on Bax and Bcl-2 levels, 7 days after Aβ_25–35 injection. Typical blots are represented above the bar graphs. Mice were injected with ANAVEX2-73 or vehicle before Aβ_25–35 peptide (9 nmol). n = 5–15 per groups. F_(4,44) = 1.98, p > 0.05 in (A) ; F_(4,44) = 1.95, p > 0.05 in (B) ; F_(4,44) = 2.66, p < 0.05 in (C); F_(4,49)= 3.01, p < 0.05 in (D), F_(4,49) = 0.89, p > 0.05 in (E), F_(4,49) = 3.09, p < 0.05 in (F). ^*p < 0.05, ^**p < 0.01 vs. Sc. Aβ; ^#p < 0.05 vs. Aβ_25–35; Dunnett’s test.