Research paperNeuron-related blood inflammatory markers as an objective evaluation tool for major depressive disorder: An exploratory pilot case-control study
Introduction
Major depressive disorder (MDD) is a common psychiatric disorder. The prevalence of MDD is considered to be 4.7% worldwide, and its burden is extremely large (DALYs and Collaborators, 2017, Ferrari et al., 2013). However, MDD diagnosis and symptom evaluation have depended on subjective information such as physician's clinical experiences and patient's self-assessments, which can unfortunately lead to misdiagnoses (Mitchell et al., 2009). Such difficulties have caused confusion in clinical practice of MDD (Kato et al., 2016, Kato and Kanba, 2017, Kato et al., 2011b, 2011c). Thus, to resolve the limitations in clinical practice of MDD, the need for biological biomarkers in the objective clinical evaluation of MDD has increased.
Previous research has revealed that neuroinflammation is very important in the pathophysiology of MDD, and that inflammatory processes may be involved with at least some of neurodegeneration in MDD (Dantzer et al., 2008, Goldsmith et al., 2016, Maes et al., 2009). Inflammatory cytokines and their receptors such as interleukin (IL) 1β, IL6, IL34, tumor necrosis factor (TNF) α, and TNF receptor 1 (TNFR1) are known to be associated with neuroinflammation linked to MDD (Chitu et al., 2016, Dowlati et al., 2010, Goldsmith et al., 2016, Grassi-Oliveira et al., 2009). In addition, postmortem brain studies in patients with depression and rodents with depression model have reported altered levels of synaptophysin (SYP), one of the synaptic vesicle phosphoproteins reflecting synaptic functions and density (Gilabert-Juan et al., 2012, Greengard et al., 1993, Liu et al., 2015). Such studies have suggested synaptic dysfunction in MDD, and portions of synaptic dysfunction may be caused by inflammatory cytokines (Sofroniew, 2014, Vezzani and Viviani, 2015). On the other hand, the orexin neuronal system has been shown to play important roles in the regulation of sleep/wakefulness, feeding behavior, reward system, and emotion, which are often disturbed in depression (Abbas et al., 2015, Chieffi et al., 2017, Sakurai, 2014, Soya et al., 2017). Orexin acts on orexin receptors (OXR), orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Interestingly, the orexin neuronal system has been also reported to modulate synaptic functions, and closely related to monoaminergic neurons (Chieffi et al., 2017, Sakurai, 2014). Therefore, the orexin neuronal system may be involved in the pathophysiology of MDD (Abbas et al., 2015, Chieffi et al., 2017, Nollet and Leman, 2013, Sakurai, 2014). Based on such evidence, we have hypothesized that the above proteins (related to neuroinflammation and synaptic functions), which are more or less carried from neurons in the brain into peripheral blood via exosomes, may be useful biomarkers for MDD (Brites and Fernandes, 2015, Chieffi et al., 2017, Greengard et al., 1993, He et al., 2015, Kandere-Grzybowska et al., 2003, Murray et al., 2015, Ogoshi et al., 2005, Ohgidani et al., 2014, Qu et al., 2007, Reinhardt et al., 2013, Sakurai, 2014, Sallmann et al., 2000, Stellwagen et al., 2005).
Various research methods including peripheral blood tests have been utilized in biomarker studies of MDD. Cerebrospinal fluid (CSF) analysis and brain imaging/brain physiological examination such as magnetic resonance imaging and electroencephalogram are known as biomarker research methods directly reflecting brain pathophysiology (Gadad et al., 2017, Gong and He, 2015, Gururajan et al., 2016, Kunugi et al., 2015). CSF-based biomarkers are invasive and brain imaging/brain physiological examination-based biomarkers are expensive and unsuitable for analysis of molecular dynamics, limiting their appeal to both patients and physicians (Mustapic et al., 2017). In contrast, a peripheral blood test can be carried out easily, quickly, and widely not only in research institutes but also primary care settings. However, peripheral blood test-based biomarkers are not necessarily expressive of molecular dynamics in the brain, since the brain and peripheral blood are strictly separated by the blood-brain barrier (Raison et al., 2010).
Recently, exosomes obtained from peripheral blood have attracted attention in various fields ranging from mechanism studies aimed at understanding the pathophysiology of various diseases such as cancers and neurodegenerative diseases to drug discovery research (Brites and Fernandes, 2015, Goh et al., 2017, Levy, 2017, Luarte et al., 2017, Mitsuhashi et al., 2013, Muller et al., 2016, Sarko and McKinney, 2017, Tavakolizadeh et al., 2017, Tsilioni et al., 2014). Exosomes are approximately 100 nm extracellular vesicles with lipid bilayer membranes, considered to be secreted from most cell types including neurons (Raposo and Stoorvogel, 2013, Yanez-Mo et al., 2015). Exosomes have been recognized as potent vehicles of intercellular communication, due to their capacity to transfer proteins, lipids, and RNA (Raposo and Stoorvogel, 2013, Yanez-Mo et al., 2015). Moreover, recent studies have reported that the composition of exosomes is different depending on cell types, states, and environments (Beninson and Fleshner, 2014, de Jong et al., 2012). In addition, since exosomes can cross the blood-brain barrier from both directions, circulating exosomes in blood are expected to reveal the pathophysiology of brain diseases without using brain biopsy and CSF analysis (Mustapic et al., 2017). Thus, peripheral blood biomarker studies of brain diseases have utilized exosomes so as to overcome the above-mentioned drawbacks of peripheral blood tests, and recent studies have reported several methods to extract neuron-derived exosomes (NDE) from peripheral blood (Fiandaca et al., 2015, Goetzl et al., 2015, Mustapic et al., 2017). On the other hand, one of the most crucial issues in current human NDE studies is that a considerable amount of peripheral blood (at least 500 µL/each patient) is required to extract exosomes, which is a limitation in measuring multiple substances contained in the NDE (Mustapic et al., 2017). Exosomes have recently been suggested to be promising in the evaluation, diagnosis, and treatment of depression, however, to our knowledge, NDE-based clinical studies focusing on depression and in particular MDD have not been reported until now (Brites and Fernandes, 2015, Tavakolizadeh et al., 2017). We have hypothesized that if blood proteins of interest are positively correlated with NDE, these proteins likely come from NDE. We have proposed that measuring the levels of neuron-related blood biomarkers normalized by the levels of NDE (quantity of each biomarker/NDE) in patients with brain diseases including MDD may be useful to predict altered levels of substances contained in the NDE without actually extracting NDE. Just recently, using a small amount of peripheral blood (less than 50 µL/each patient), we have established a novel sandwich immunoassay between anti-neuron antibody (synaptosome associated protein 25 (SNAP25), a neuronal protein involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane (Sadoul et al., 1995)) and antibodies against CD81 (one of the typical markers of exosomes) and against other targeting proteins (Fig. 1) (Kawata et al., 2018). Using this method, we have revealed that the levels of neuron-related blood targeting proteins normalized by the levels of CD81 (NDE) are significantly associated with the clinical concussion prognosis and recovery process in patients with sport-related neurotrauma (Kawata et al., 2018).
Based on the above-introduced hypothesis of depression, we have suggested that some proteins related to neuroinflammation and synaptic functions may be carried from neurons in the brain into peripheral blood via exosomes during the clinical course of depression, and that measuring such neuron-related blood proteins normalized by CD81 (NDE) may be useful for the objective clinical evaluation of MDD. Thus, as an exploratory pilot case-control study between healthy controls (HC) and drug-free MDD patients, we herein searched for NDE-related blood biomarkers using the above sandwich immunoassay with a small amount of peripheral blood (less than 50 µL/each patient).
Section snippets
Methods
The present study was approved by the ethics committee of Kyushu University and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.
Results
Demographics and clinical variables were described in Table 1. Drug-free MDD patients (N = 34) were in acute episode of MDD (disease duration: 4.0 (4.0) weeks). SOD of most patients was between moderate to severe in accordance with BDI-II total score (29.9 ± 11.1). There were no significant differences in sex and age between HC and MDD group.
Discussion
The present exploratory pilot study suggests, for the first time, the clinical utility of neuron-related blood biomarkers normalized by CD81 (NDE) in drug-free MDD patients, using a novel ELISA method with a small amount of peripheral blood. First, this study has shown that most neuron-related blood biomarkers including IL34 and TNFR1 have moderately to strongly positive correlation with CD81 (NDE). Based on such significant correlation between neuron-related blood biomarkers and CD81, we
Limitations
The present exploratory pilot study has some limitations. First, we did not actually extract NDE from peripheral blood to measure the biomarkers contained in the NDE. It is important to note that one of the most crucial problems in current human NDE studies is that a considerable amount of peripheral blood is required to extract exosomes from peripheral blood and to measure multiple substances contained in the NDE. Fortunately, using a small amount of peripheral blood, we herein successfully
Conclusions
In the present blood biomarker study, we have shown the clinical utility of neuron-related blood biomarkers normalized by CD81 (NDE) in drug-free MDD patients, using a novel ELISA method with a small amount of peripheral blood. The present study has highlighted the important interactions between neuroinflammation, neuronal damage, and synaptic dysfunction possibly associated with inflammatory cytokines in the pathophysiology of MDD at least during the acute phase. Diagnosis and symptoms
Conflicts of interest statement
All the authors have declared that no conflicts of interest exist.
Role of funding source
This work was partially supported by Grant-in-Aid for Scientific Research on (1) Innovative Areas “Will-Dynamics” and "Glia Assembly" of The Ministry of Education, Culture, Sports, Science, and Technology, Japan (JP16H06403 to T.A.K.; JP25117011 to S.K.), (2) The Japan Agency for Medical Research and Development (AMED). (Syogaisya-Taisaku-Sogo-Kenkyu-Kaihatsu-Jigyo to T.A.K. & S.K. (JP17dk0307047, & JP18dk0307075), and Yugo-No to T.A.K. (JP18dm0107095)), (3) KAKENHI - the Japan Society for the
Contributors
T.A.K., M.M., and N.K. contributed to the conception and design.
T.A.K. was responsible for protocol of the study.
N.K., T.A.K., M.M., M.S-K., N.Sh., K.H., M.O., N.Sa., and H.K. contributed to the investigation.
N.K., T.A.K., and M.M. contributed to the data checking, analysis, and interpretation of data.
N.K. and T.A.K. drafted the article, and T.S. and S.K. revised it critically for important intellectual content.
All the authors provided final approval of the version to be published.
Acknowledgments
The authors would like to thank Dr. Koji Tanaka, Dr. Junji Kishimoto, Mr. Keita Kurahara, Ms. Ryoko Katsuki, Ms. Sakumi Kakimoto, and Ms. Yoko Zushi for their research support and/or assistance.
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