Parishin A ameliorates cognitive decline by promoting PS1 autophagy in Alzheimer's disease

Abstract

Introduction: Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. Its pathological features include: A lot of misfolding and abnormal aggregation of amyloid protein (Aβ); Autophagy disorder, oxidative stress, neuroinflammation, abnormal phosphorylated tau protein and synaptic dysfunction. Modern pharmacological studies have found that Paisinhin A (PA) has beneficial effects on the prevention and treatment of central nervous system diseases. This study aims to explore the role and mechanism of PA in AD through autophagy pathway, and lay a scientific foundation for the development of clinical prevention and treatment strategies for AD.

Methods: N2A^APP cells were treated with different concentrations of PA. Cell viability was detected by CCK-8 method. Western blotting detected the expression levels of proteins related to amyloid production, autophagy pathway, and phosphorylated Tau expression levels. Autophagy flow was detected by transfecting Lc3 double fluorescent plasmid. After Aβ was injected into the hippocampus of WT mice and PA was injected intraperitoneally, the learning and memory ability of WT mice were tested by new object recognition, y maze and water maze. The oxidative stress level was detected by the kit. The levels of inflammatory factors were detected by RT-qpcr.

Results: The viability of N2A^APP cells was not affected at different concentrations of PA, but PS1 was significantly decreased at 40μM. PA can obviously improve the accumulation of autophagy in AD, and to some extent save the autophagy inhibition of CQ. Behavioral studies have shown that PA can also improve learning and memory impairments caused by Aβ injections. In addition, in vivo experiments, PA can also improve oxidative stress levels, inflammation levels and salvage dysfunctions of synapses. PA also reduces the levels of total and phosphorylated Tau in N2A^Tau.

Discussion: Our study provides the first evidence that PA improves learning and memory in Aβ-induced AD mice. This effect appears to be mediated by PA by promoting autophagy and reducing oxidative stress. It was also found that PA may have a role in regulating inflammation, improving abnormally phosphorylated tau, and salvaging damaged synaptic function, providing valuable insights into potential applications in the treatment and prevention of AD.

Keywords: Alzheimer’s disease; PS1; Parishin A; amyloid-β; autophagy.

Figure 1

PA inhibits APP amyloidogenic processing by targeting PS1 in N2A^APP cells. (A) The cell viability assessed by CCK-8 in N2A^APP cells treated with a gradient concentration of PA (0–320 μM) for 24 h, n = 4 per group. (B–E) Protein levels of PS1 (B,C), APP (B,D), and BACE1 (B,E) assessed by Western blotting in N2A^APP cells were treated with gradient concentrations of PA (0–320 μM) for 24 h, n = 3 per group. Data are presented as mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

PA promotes autophagy-lysosomal degradation of PS1. (A) PS1 mRNA levels assessed by qRT-PCR in N2A^APP cells treated with PA (40 μM, 24 h). n = 4 in each group. (B,C) Effect of PA on PS1 degradation assessed by half-life measurements in N2A^APP cells treated with 100 μg/mL cycloheximide (CHX). n = 5–6 in each group. (D) The total ubiquitination level and the ubiquitination level of PS1 assessed by Western blot and Co-IP in N2A^APP cells treated with PA (40 μM for 24 h). n = 2 in each group. (E,F) The protein level of PS1 assessed by Western blot in N2A^APP cells treated with PA (40 μM for 24 h) along with or without 6 h of pretreatment with CQ (50 μM for 24 h) and MG132mg (5 μM for 24 h). n = 5 in each group. Data are presented as mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Autophagy accumulation occurs in AD. (A–C) The protein levels of P62 (A,B), and LC3 (A,C) assessed by Western blot inN2A and N2A^APP cells. (D,E) The autophagic flux assessed by a fluorescence assay with mCherry-GFP-LC3 in N2A^APP cells. n = 15–25 in each group. Data are presented as mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

PA enhances the autophagy flux in N2A^APP cells. (A–C) The protein levels of P62 (A,B), and LC3 (A,C) assessed by Western blot in N2A^APP cells treated with PA (40 μM for 24 h). n = 5–6 in each group. (D–G) The protein levels of P62 (D,E), and LC3 (D,F,G) assessed by Western blot in N2A^APP cells pretreated with PA (40 μM for 6 h) and then co-treated with CQ for 24 h (50 μM). n = 4 in each group. (H,I) The autophagic flux assessed by a fluorescence assay with mCherry-GFP-LC3 in N2A^APP cells pretreated with PA (40 μM for 6 h) and then co-treated with CQ for 24 h (50 μM). n = 12–21 in each group. Data are presented as mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

PA attenuates Aβ_1–42-induced spatial learning and memory deficits in mice. (A) Flow chart of animal experiments. (B–F) Spatial learning and memory retrieval assessed by Morris water maze test in WT and Aβ-treated mice treated with or without PA treatment. The average speed of travel in the Morris water maze during the adaptation phase (B), the escape latency to the hidden platform a during the spatial learning period (C), first entry into the platform area and the number of entries into the platform zone during the memory retrieval test. n = 12–18 in each group. (G,H) The cognitive function of mice was evaluated by the novel object recognition test, and the green area was the cognitive coefficient of the novel object recognition area, (H) cognitive index; (I,J) Spontaneous alter country behavior of mice was assessed by Y-maze, and (J) spontaneous alteration. Data representation of average ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

PA reduces oxidative stress in Aβ_1–42-induced mouse model of AD. (A–C) Pro-oxidant H₂O₂ (A), i-NOS (B), and T-NOS (C) assessed by relevant commercial kits in the brains of Aβ_1–42 treated mice with or without PA treatment. n = 12–18 in each group. (D,E) antioxidants GR (D) and T- AOC (E) assessed by the relevant commercial kits in the brains of Aβ_1–42 treated mice with or without PA treatment. n = 7–13 in each group. PA regulates the levels of inflammatory cytokines in Aβ_1–42-induced mouse. The levels of inflammatory factors IL-4 (F), TNF-α (G) and IL-6 (H) in the brain of mice were treated with Aβ, and the changes were evaluated with or without PA by QRT-PCR. Each group is n = 5. PA can regulate the level of SYP protein in Aβ_1–42-induced mouse (I). The protein level of SYP (J), and PSD95 (K) assessed by Western blot in Aβ_1–42-induced mouse n = 5 each group. Data are presented as mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

A regulated protein levels of total tau and phosphotau in N2A^Taucell lines but not in Aβ_1–42-induced mouse. The protein level of Tau5 (B), Ser396 (C), Thr181 (D), Ser404 (E) and Ser199 (F) assessed by Western blot in Aβ_1–42-induced mouse n = 5 each group. The protein level of Tau5 (H) assessed by Western blot in N2A^Taucell. n = 3 in each group. The protein level of Tau5 (J), Ser199 (K), Ser396 (L), and Ser404 (M) assessed by Western blot in N2A^Taucell. n = 5–6 in each group. Data representation of average ± SEM, Aβ_1–42 Levels of total tau and phosphotau in mice induced by Aβ_1–42 after PA treatment (A); Levels of total tauin N2A^Taucells (G); Levels of total tau and phosphotau in N2A^Taucells after PA treatment (I). *p < 0.05, **p < 0.01, ***p < 0.001.