Effect of p62 on tau hyperphosphorylation in a rat model of Alzheimer's disease

Abstract

Tau hyperphosphorylation is a main cause of neuronal loss in Alzheimer's disease, which can be caused by many factors, including oxidative stress. The multifunctional protein p62, which exists in neurofibrillary tangles and causes aggregation of hyperphosphorylated tau, not only serves as a receptor in selective autophagy, but also regulates oxidative stress. However, whether p62 participates in oxidative stress-induced tau hyperphosphorylation remains unclear. In this study, we produced an Alzheimer's disease rat model by injecting β-amyloid protein into the hippocampus and β-galactose intraperitoneally. Hematoxylin-eosin staining was used for morphological analysis of brain tissue, and western blotting, immunohistochemistry and reverse transcription-PCR were employed to study p62 and autophagy related proteins, antioxidant defense system kelch-like ECH-associated protein 1-NF-E2-related factor 2 related proteins and hyperphosphorylated tau, respectively. The number of neurons in the brain decreased in Alzheimer's disease rats, and the autophagy related proteins Atg12-Atg5, microtubule-associated protein 1 light chain 3-phosphatidylethanolamine and Beclin1 increased significantly, while p62 expression reduced. Expression of kelch-like ECH-associated protein 1 increased, NF-E2-related factor 2 protein and the downstream gene products of glutamate cysteine ligase catalytic subunit and glutamate cysteine ligase modulatory subunit decreased, and hyperphosphorylated tau increased. These findings demonstrate that autophagy levels increased and p62 levels decreased in the brains of Alzheimer's disease rats. Moreover, the anti-oxidative capability of the NF-E2-related factor 2-antioxidant response element pathway was decreased, which may be the cause of tau hyperphosphorylation in Alzheimer's disease brain tissue and the subsequent structural and functional damage to neurons.

Keywords: Alzheimer's disease; NF-E2-related factor 2; autophagy; neural regeneration; p62; tau hyperphosphorylation.

Figure 1

Morphological changes in the hippocampal CA3 area and forebrain (hematoxylin-eosin staining). Scale bar: 20 μm. (A) Hippocampal CA3 area indicated in the square. (B–D) The hippocampus in the control, saline, and Alzheimer's disease (AD) model groups, respectively. (B, C) Neurons were arranged closely, had a circular shape, nuclear membrane was clear in the control and saline groups; (D) the neurons had irregular shapes and the nuclear membrane was indistinct in the AD model group. (E) Forebrain cortex indicated in the square. (F–H) The forebrain cortex in the control, saline, and AD model groups, respectively. (F, G) Neurons had a circular shape, the nuclear membrane was clear in the control and saline groups; (H) neurons had irregular shapes and the nuclear membrane was indistinct in the AD model group.

Figure 2

Changes in autophagy-associated proteins and p62 expression in the rat cerebral cortex and hippocampus. Western blot analysis (A) and quantification of the expression of Atg12-Atg5, LC3-II, beclin1 and p62 in the cerebral cortex of the saline and AD model group as compared with the control (CON) group (adjusted as 1.0) (B–E). Western blot analysis (F) and quantification of the expression of Atg12-Atg5, LC3-II, beclin1, and p62 in the hippocampus as compared with the control group (adjusted as 1.0) (G–J). β-actin was used as an internal standard. The assay was performed three times and data were expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, vs. the control group (one-way analysis of variance followed by Dunnett's t-test). Atg: Autophagy-related genes; LC3: microtubule-associated protein 1 light chain 3; AD: Alzheimer's disease.

Figure 3

Changes in Keap1 and Nrf2 expression in the rat cerebral cortex and hippocampus. (A, B) Immunohistochemical and quantitative analysis for the expression of Keap1 in the cerebral cortex of Alzheimer's disease (AD) model rats. The expression of Keap1 in AD model rats was increased compared with the control (CON) group. (C, D) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the cerebral cortex of AD model rats. The expression of Nrf2 in AD model rats increased compared with control rats. (E, F) Immunohistochemical and quantitative analysis for the expression of Keap1 in the hippocampus of AD model rats. The expression of Keap1 in AD model rats increased compared with control rats. (G, H) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the hippocampus of AD model rats. The expression of Nrf2 in AD model rats remained unchanged compared with control rats. Scale bars: 20 μm. ^aP < 0.05, vs. control rats (one-way analysis of variance followed by Dunnett's t-test). The assay was performed three times and data were expressed as mean ± SD. Nrf2: NF-E2-related factor 2; Keap1: kelch-like ECH-associated protein 1.

Figure 4

Changes in glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM) mRNA expression in the rat cerebral cortex and hippocampus. (A) RT-PCR analysis for mRNA expression of GCLM and GCLC in the cerebral cortex; (B, C) quantification of GCLM and GCLC mRNA expression in the cerebral cortex compared with the control (CON) group (adjusted as 1.0). (D) Reverse transcription-PCR analysis for mRNA expression of GCLM and GCLC in the hippocampus; (E, F) quantification of GCLM and GCLC mRNA expression in the hippocampus compared with the control group (adjusted as 1.0). GAPDH was used as an internal standard. ^aP < 0.05, ^bP < 0.01, vs. control group (one-way analysis of variance followed by Dunnett's t-test). The assay was performed three times and data were expressed as mean ± SD.

Figure 5

Changes in tau and p-tau protein expression in the rat cerebral cortex and hippocampus. (A) Western blot analysis for the expression of total tau and p-tau in the cerebral cortex; (B, C) quantification of tau and p-tau protein expression in the cerebral cortex compared with the control (CON) group (adjusted as 1.0). (D) Western blot analysis for the expression of total tau and p-tau protein in the hippocampus; (E, F) quantification of tau and p-tau protein expression in the hippocampus compared with the control group (adjusted as 1.0). β-actin was used as an internal standard. ^aP < 0.05, vs. the control group (one-way analysis of variance followed by Dunnett's t-test). The assay was performed three times and data were expressed as mean ± SD.