Research reportProbing amyloid beta-induced cell death using a fluorescence-peptide conjugate in Alzheimer's disease mouse model
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.
References (38)
- et al.
In vivo imaging of myocardial cell death using a peptide probe and assessment of long-term heart function
J. Control. Release
(2013) - et al.
Inflammatory mediator and beta-amyloid (25-35)-induced ceramide generation and iNOS expression are inhibited by vitamin E
Free Radic. Biol. Med.
(2004) - et al.
CSF markers for incipient Alzheimer's disease
Lancet Neurol.
(2003) - et al.
Nanotechnologies for Alzheimer's disease: diagnosis, therapy, and safety issues
Nanomedicine
(2011) - et al.
In vivo oxidative stress in brain of Alzheimer disease transgenic mice: requirement for methionine 35 in amyloid beta-peptide of APP
Free Radic. Biol. Med.
(2010) - et al.
Discrimination between Alzheimer dementia and controls by automated analysis of multicenter FDG PET
NeuroImage
(2002) - et al.
Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial
Lancet
(2008) - et al.
Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes
Brain Res.
(2004) - et al.
Fibrillar amyloid-beta peptides kill human primary neurons via NADPH oxidase-mediated activation of neutral sphingomyelinase. Implications for Alzheimer's disease
J. Biol. Chem.
(2004) - et al.
Protective effects of S-nitrosoglutathione against amyloid beta-peptide neurotoxicity
Free Radic. Biol. Med.
(2005)
The therapeutic potential of human umbilical cord blood-derived mesenchymal stem cells in Alzheimer's disease
Neurosci. Lett.
Bone marrow-derived mesenchymal stem cells reduce brain amyloid-beta deposition and accelerate the activation of microglia in an acutely induced Alzheimer's disease mouse model
Neurosci. Lett.
Topographic associations between DNA fragmentation and Alzheimer's disease neuropathology in the hippocampus
Neurochem. Int.
In vivo imaging of tumor apoptosis using histone H1-targeting peptide
J. Control. Release
3D comparison of hippocampal atrophy in amnestic mild cognitive impairment and Alzheimer's disease
Brain
Apoptosis and Alzheimer's disease
J. Neural Transm.
Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds
Biol. Chem.
Cognitive and brain profiles associated with current neuroimaging biomarkers of preclinical Alzheimer's disease
J. Neurosci.
Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination
J. Neurosci. Res.
Cited by (6)
Imaging Cell Death: Focus on Early Evaluation of Tumor Response to Therapy
2020, Bioconjugate ChemistryDecreased netrin-1 and correlated Th17/Tregs balance disorder in Aβ<inf>1-42</inf> induced Alzheimer's disease model rats
2019, Frontiers in Aging NeuroscienceAmino acid and peptide bioconjugates
2018, Amino Acids, Peptides and Proteins
- 1
These authors contributed equally to this work.