Pharmacological inhibition of asparaginyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model

Abstract

Background: Currently, there is no cure for Alzheimer's disease (AD). Therapeutics that can modify the early stage of AD are urgently needed. Recent studies have shown that the pathogenesis of AD is closely regulated by an endo/lysosomal asparaginyl endopeptidase (AEP). Inhibition of AEP has been reported to prevent neural degeneration in transgenic mouse models of AD. However, more than 90% of AD cases are age-related sporadic AD rather than hereditary AD. The therapeutic efficacy of AEP inhibition in ageing-associated sporadic AD remains unknown.

Methods: The senescence-accelerated mouse prone 8 (SAMP8) was chosen as an approximate model of sporadic AD and treated with a selective AEP inhibitor,: δ-secretase inhibitor 11. Activation of AEP was determined by enzymatic activity assay. Concentration of soluble amyloid β (Aβ) in the brain was determined by ELISA. Morris water maze test was performed to assess the learning and memory-related cognitive ability. Pathological changes in the brain were explored by morphological and western blot analyses.

Results: The enzymatic activity of AEP in the SAMP8 mouse brain was significantly higher than that in the age-matched SAMR1 mice. The half maximal inhibitory concentration (IC₅₀) for δ-secretase inhibitor 11 to inhibit AEP in vitro is was around 150 nM. Chronic treatment with δ-secretase inhibitor 11 markedly decreased the brain AEP activity, reduced the generation of Aβ_1-40/42 and ameliorated memory loss. The inhibition of AEP with this reagent not only reduced the AEP-cleaved tau fragments and tau hyperphosphorylation, but also attenuated neuroinflammation in the form of microglial activation. Moreover, treatment with δ-secretase inhibitor 11 prevented the synaptic loss and alleviated dendritic disruption in SAMP8 mouse brain.

Conclusions: Pharmacological inhibition of AEP can intervene and prevent AD-like pathological progress in the model of sporadic AD. The up-regulated AEP in the brain could be a promising target for early treatment of AD. The δ-secretase inhibitor 11 can be used as a lead compound for translational development of AD treatment.

Keywords: Alzheimer’s disease; Asparaginyl endopeptidase; Legumain; SAMP8 mouse; Therapeutic target; δ-Secretase inhibitor 11.

Fig. 1

Protein expression and activity of AEP in brains of SAMP8 and SAMR1 mice. a Western blotting bands of mature AEP in the hippocampus and cortex of 4-month-old SAMP8 mice and age-matched SAMR1 mice. b Activity of AEP in the whole brains of 4-month-old SAMP8 and SAMR1 mice. * P < 0.05, Student’s t-test, n = 5 per group. c Mature AEP in the hippocampus and cortex of 6-month-old SAMP8 mice and age-matched SAMR1 mice. d AEP activity in the whole brains of 6-month-old SAMP8 and SAMR1 mice. * P < 0.05, unpaired Student’s t-test, n = 5 per group

Fig. 2

Pharmacological action of the δ-secretase inhibitor 11. a The enzymatic activity of AEP was measured by a 45-min fluorescent substrate cleavage assay. b The concentration-response of δ-secretase inhibitor 11 on AEP activity. Data were obtained at 45 min after the substrate cleavage reaction, n = 5 independent experiments. c The enzymatic activity of AEP in brains of SAMP8 and SAMR1 mice was measured by a 45-min fluorescent substrate cleavage assay. d AEP activity was obtained at 45 min after the substrate cleavage reaction and compared between groups. *P < 0.05, n = 6 mice per group, one-way ANOVA followed by a Tukey’s post-hoc test. e, f Measurement of Aβ_1–40 and Aβ_1–42 in the brains of SAMP8 mice treated with vehicle or δ-secretase inhibitor 11. *P < 0.05, unpaired Student’s t-test; n = 6 mice per group

Fig. 3

Effect of δ-secretase inhibitor 11 treatment on memory function of mice. a, b The swimming speed and escape latency of mice over the 5-day acquisition training. Two-way ANOVA analysis revealed the time effect (F (2.922, 35.06) = 9.659, P < 0.001) and group effect (F (3, 12) = 7.355, P < 0.05) on escape latency. SAMP8 mice learned significantly slower than SAMR1 mice (P < 0.05), and the vehicle-treated SAMP8 mice learned significantly slower than the δ-secretase inhibitor 11-treated SAMP8 mice (P < 0.05). n = 9 mice per group. c, d The percentage of time spent and the distance travelled in the target quadrant in the probe test, which was performed on day 6. *P < 0.05, n = 9 mice per group, one-way ANOVA followed by a Tukey’s post-hoc test

Fig. 4

Treatment with δ-secretase inhibitor 11 reduced pathological changes of tau in SAMP8 mice. a, b Western blot analysis of AEP and tau N368. *P < 0.05, unpaired Student’s t-test; n = 4 mice per group. c Immunostaining of phospho-tau (Thr231) in cortical and hippocampal neurons. d, e Density of phospho-tau-positive cells in the cortex and hippocampus of SAMP8 mice. *P < 0.05 between groups, unpaired Student’s t-test; n = 5 mice per group

Fig. 5

Treatment with δ-secretase inhibitor 11 reduced pathological activation of microglia in SAMP8 mice. a Immunostaining of Iba1, a marker of microglial activation, in the cortex and hippocampus. b, c Density of Iba1-positive cells in the cortex and hippocampus of SAMP8 mice. *P < 0.05, between groups. n = 5 mice per group, unpaired Student’s t-test. d Western blot analysis of Iba1. *P < 0.05, unpaired Student’s t-test; n = 6 mice per group

Fig. 6

Treatment with δ-secretase inhibitor 11 restored MAP-2 and synapse-associated protein expression in SAMP8 mice. a Immunostaining of the neuronal marker MAP-2 in the cortex and hippocampus. Arrows indicate neuronal soma and dendrites stained by MAP-2. b, c MAP-2 immunoreactivity was illustrated as the percent area of the neuropil in the cortex and hippocampus of SAMP8 mice. *P < 0.05, unpaired Student’s t-test, n = 5 mice per group. d, e Western blot analysis of brain lysate showing the expression of postsynaptic density protein 95 (PSD-95) and synaptophysin (SYP) in the two groups. *P < 0.05 between groups, unpaired Student’s t-test; n = 6 mice per group