Elsevier

Brain Research

Volume 1646, 1 September 2016, Pages 514-521
Brain Research

Research report
Probing amyloid beta-induced cell death using a fluorescence-peptide conjugate in Alzheimer's disease mouse model

https://doi.org/10.1016/j.brainres.2016.06.041Get rights and content

Highlights

  • Apoptosis caused by Aβ has been shown to play an important role in AD pathology.

  • The ApoPep-1 co-localized with brain cells which undergo apoptosis in AD mice.

  • ApoPep-1 could be an assistant tool for molecular imaging in AD diagnosis.

Abstract

With the increasing worldwide incidence of Alzheimer's disease (AD), there is a critical need for the discovery of more effective diagnostic methods. However, development of diagnostic tools in AD has been hindered by obstacles such as the absence of exact biomarkers. Apoptosis caused by amyloid-β (Aβ) plays an important role in AD pathology; therefore, provides an attractive biological target for the diagnosis of AD. The present study aimed to evaluate the potential of small peptide, named ApoPep-1 (Apoptosis-targeting peptide-1) as a new apoptosis imaging agent in AD. The fluorescein-conjugated ApoPep-1, but not the control peptide, targeted apoptotic cells in the brain of amyloid precursor protein (APP)/presenilin 1 (PS1) mice. We also observed fluorescence signals during in vivo imaging of apoptotic cells using ApoPep-1, and fluorescence levels increased in an age-dependent manner in APP/PS1 mice. Ex vivo imaging of isolated brains in APP/PS1 mice further confirmed the targeting of ApoPep-1 to apoptotic cells. The fluorescein-labeled ApoPep-1 co-localized with brain cells such as neurons, astrocytes, and microglia, all of which undergo apoptosis in the APP/PS1 mice brain. These findings demonstrate that ApoPep-1 can target apoptotic brain cells, and be used for experimental investigations relevant to apoptosis in AD.

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia in the elderly. This neuropathological condition is characterized by a progressive loss of cognitive function, and is defined by two established pathophysiological hallmarks in the brain. These are extracellular accumulations composed primarily of the amyloid-β (Aβ) peptide, and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein that promote neuronal apoptosis (Hardy and Selkoe, 2002, Querfurth and LaFerla, 2010).

Millions of people are currently affected by this disease. It is predicted that within the next few decades, AD will exert a huge social and economic impact if no efficient therapeutics and/or early diagnosis approaches become available (Brambilla et al., 2011). Therefore, an important challenge in the management of AD is to establish a method for effective diagnosis, in order to identify patients with AD prior to the actual onset of dementia (Forlenza et al., 2010). Due to the serious clinical need to predict adverse outcomes in patients at high risk of AD, there have been many developments in AD biomarker research. These include the development of cerebrospinal fluid (CSF) biomarkers (Blennow and Hampel, 2003, Clark et al., 2003, Buerger et al., 2006) and structural/functional neuroimaging protocols (Herholz et al., 2002, Singh et al., 2006). However, some limitations need to be overcome before these tools can be introduced into clinical practice. For example, fluid biomarkers are needed assay standardization and anatomical precision in the measurements. Magnetic resonance imaging and positron emission tomography are relatively expensive and require experienced personnel. Therefore, there is still a need for better diagnostic approaches to detect AD. Recently, although optical imaging by peptide-probe has the limitation of depth in tissue penetration, it is one of the most widely used imaging modality in clinical practice and in research. Compared to other imaging systems, optical imaging has many advantages, as it enables non-invasive, and safe detection using readily available instruments at moderate cost. Also, due to their advantages of high sensitivity, optical imaging plays a central role in the investigation of disease diagnosis and relevant drug development (Edgington et al., 2009).

Apoptosis caused by Aβ has been shown to play an important role in AD pathology. Although some researchers have suggested that there is a poor correlation between amyloid plaque load and the presence of dementia in AD (Engler et al., 2006, Holmes et al., 2008), many studies indicate that Aβ triggers a cascade of pathogenic events that culminate in neuronal apoptosis/death, neuritic dystrophy, and oxidative stress (Behl and Moosmann, 2002, Butterfield et al., 2010, Yang et al., 2009). Therefore, cellular apoptosis driven by Aβ provides an attractive biological target to better predict AD in individual patients. Although peptide-based probes have brief serum half-lives caused by degradation or excretion, small peptides as imaging probes have many potential advantages including more efficient penetration into tissues, easier conjugation with imaging agents and higher specificity to targets compared to proteins and antibodies. Additionally, small peptides have a low production cost and low immunogenicity (Lee et al., 2010). ApoPep-1 (Apoptosis-targeting peptide-1), six-amino-acid CQRPPR peptide, recognizes apoptotic and necrotic cells by binding to histone H1 exposed on the cell surface and located at the nucleus (Wang et al., 2010). ApoPep-1 has been successfully used for in vivo imaging of cell death in tumor cells and myocardial cells (Wang et al., 2010, Acharya et al., 2013).

The present study was designed to evaluate the feasibility of ApoPep-1, as an imaging tool for apoptosis in AD. Here, we have shown that ApoPep-1 bound to primary cultured apoptotic cells under conditions simulating AD, as well as to apoptotic brain cells from AD mice. Our findings demonstrate that ApoPep-1 could be an effective tool for molecular imaging of apoptosis in AD animals.

Section snippets

In vitro binding of ApoPep-1 to apoptotic brain cells

To investigate the binding of ApoPep-1 to apoptotic brain cells, we cultured primary neurons, astrocytes, and microglia (Fig. S1). The experimental protocol is described in Fig. 1A. To induce cell death, primary cultured brain cells were treated with Aβ42 for 24 h. Similar to previous studies (Lee et al., 2010a, Lee et al., 2010b), aggregated Aβ induced apoptosis in neurons (Fig. 1B). The aggregated Aβ42 also evoked apoptosis in primary cultured microglia and astrocytes. The signals of ApoPep-1

Discussion

In this study, we evaluated whether ApoPep-1 could be used as a new apoptosis imaging agent in an AD animal model. By obtaining optical imaging of the ApoPep-1 signal, both in vivo and ex vivo, we observed that ApoPep-1 could detect apoptotic cell death in brain tissue in an AD model. In addition, we show that ApoPep-1 signals were elevated in AD mice related to the increase of apoptosis with disease progression. Based on these results, we suggest that ApoPep-1 could be an effective tracer for

Primary cell culture

Hippocampal neurons from embryonic day (E)18 C57BL/6 mice were prepared as described previously, with minor modifications (Brewer et al., 1993). Hippocampi were dissected and dissociated, followed by incubation in papain (Worthington) for 15 min at 37 °C. Neurons were plated on poly-l-lysine (Sigma-Aldrich) coated coverslips at 37 °C in a humidified atmosphere with 5% CO2. After cells had attached to the substrate, the medium was replaced with neuronal culture medium; specifically, serum-free

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the Basic Science Research Program (2014R1A2A1A10051107 and 2015R1A2A1A01004779) of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea. This study was also supported by the NRF grant funded by the Korea government (2014R1A5A2009242). Additional support for this work was provided by the KIST Institutional Program (Project no. 2E25000), Republic of Korea.

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