Brain Research

Volume 1821, 15 December 2023, 148565
Brain Research

Injection of exogenous amyloid-β oligomers aggravated cognitive deficits, and activated necroptosis, in APP23 transgenic mice

https://doi.org/10.1016/j.brainres.2023.148565Get rights and content

Highlights

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    Stereotaxic injection of AβO into the brain of APP23 transgenic mice to establish a novel AD mouse model.
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    Study the effect of AβO on memory impairment, AβO accumulation, tau protein phosphorylation level.
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    Necroptosis induced by AβO injection occurred predominantly in microglia of the AD brain.
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    Possibility of necroptosis as a potential intervention and therapeutic target for AD.

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by the loss of synapses and neurons in the brain, and the accumulation of amyloid plaques. Aβ oligomers (AβO) play a critical role in the pathogenesis of AD. Although there is increasing evidence to support the involvement of necroptosis in the pathogenesis of AD, the exact mechanism remains elusive. In the present study, we explored the effect of exogenous AβO injection on cell necroptosis and cognitive deficits in APP23 transgenic mice. We found that intrahippocampal injection of AβO accelerated the development of AD pathology and caused cognitive impairment in APP23 mice. Specifically, AβO injection significantly accelerated the accumulation of AβO and increased the expression level of phosphorylated-tau, and also induced necroptosis. Behavioral tests showed that AβO injection was associated with cognitive impairment. Furthermore, necroptosis induced by AβO injection occurred predominantly in microglia of the AD brain. We speculate that AβO increased necroptosis by activating microglia, resulting in cognitive deficits. Our results may aid in an understanding of the role played by AβO in AD from an alternative perspective and provide new ideas and evidence for necroptosis as a potential intervention and therapeutic target for AD.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by the loss of synapses and neurons in the brain and the accumulation of amyloid plaques (Long and Holtzman, 2019, Hillen, 2019). Amyloid beta (Aβ), which is a proteolytic derivative of amyloid precursor protein (APP), plays a critical role in the pathogenesis of AD (Westermark et al., 2007, Chen et al., 2017). Both excessive production and impaired clearance of Aβ can lead to the accumulation of Aβ plaques in the brain (Levin and Vasenina, 2016, Jack et al., 2018).
Soluble Aβ oligomers (AβO) are a synaptotoxic form of Aβ associated with AD pathogenesis (Savage et al., 2014, Yang et al., 2017, Hong et al., 2018). The level of AβO isolated from the brains of AD patients was strongly correlated with the onset of AD progression and the severity of clinical symptoms (Fukumoto et al., 2010, Kasai et al., 2017). In the brains of AD patients, AβO penetrates cell membranes and causes a variety of pathological changes, including oxidative stress, mitochondrial dysfunction, synaptic defects, and abnormal tau protein changes, leading to cognitive impairment (Prasansuklab and Tencomnao, 2013, Forloni et al., 2016, Bode et al., 2017, John and Reddy, 2021, Araki and Kametani, 2022).
Severe neuronal loss is a hallmark of AD, but the mechanism of neuronal death remains elusive. Necroptosis is a regulated form of necrotic cell death that can be activated under conditions of defective apoptosis (Degterev et al., 2005, Salvadores and Court, 2020), and several studies found that the expression of necroptotic markers was correlated with the progression of AD stages and cognitive levels (Caccamo et al., 2017, Koper et al., 2020). Experimentally, necrotrophic apoptosis damaged neurons in mouse models, leading to cognitive dysfunction (Caccamo et al., 2017). Cell necroptosis is thus a possible pathway related with the progression of AD (Zhang et al., 2022, Richard and Mousa, 2022).
Despite extensive basic research on the pathogenesis of AD, translating the results of this research into clinical treatment is still challenging. Therefore, a clearer understanding that would explain how Aβ ccumulates in the brain of AD patients is necessary. In our current study, we developed a new AD mouse model via stereotactic injection of AβO into the brains of APP23 transgenic mice. The objective was to determine the impact of AβO injection on several factors including memory impairment, tau protein phosphorylation levels, AβO accumulation, and necroptosis activation.

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Section snippets

Dot blotting analysis of AβO

Dot blotting analysis was used to verify the preparation of AβO results. Increased immunoreactivity to A11 antibody was observed after AβO preparation (Fig. 1a and b, p < 0.0001).

8-arm maze behavior test

In the working memory and reference memory behavioral tests with the 8-arm radial maze, there was no significant difference between the WT and APP23 groups at 2 months before the injection (Fig. 2a, p = 0.9794; 2b, p = 0.4968; 2e, p = 0.9312; 2f, p = 0.2277). On the other hand, the APP23 + AβO group showed

Discussion

The present study is the first report to evaluate the effect of intrahippocampal injection of AβO on cognitive deficits in the APP23 transgenic AD mice model. The two principal findings of this study were: 1) injection of AβO in APP23 mice significantly impaired cognitive impairment in mice (Fig. 2); 2) mice developed cognitive dysfunction when they were 6 months old (1 month after injection), and age when mice do not normally exhibit cognitive dysfunction. We also observed the acceleration and

AβO preparation

To obtain a 5 mM concentration of AβO, 1 mg of recombinant human Aβ1-42 (#AG968, Sigma-Aldrich, MO, USA) was dissolved in 44 μL of fresh anhydrous dimethyl sulfoxide (DMSO, #D5879, Sigma-Aldrich). This solution was ultrasonicated for 1 min, then added to ice-cold DMEM/F-12 medium (#11039047, Gibco, Grand Island, UK) to achieve a final concentration of 100 μM. This solution was incubated at 4 °C for 24 h and then centrifuged at 14,000 × g for 10 min. The supernatant was transferred to a new tube

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was partly supported by a Grant-in-Aid for Scientific Research (C) 20 K09370, 20 K12044, Challenging Research 21 K19572, Young Research 20 K19666, 21 K15190, and by Grants-in-Aid from the Research Committees (Toba K, and Tsuji S) from the Japan Agency for Medical Research and Development.

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