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Journal of Alzheimer’s Disease 39 (2014) 253–259 DOI 10.3233/JAD-130932 IOS Press
MicroRNAs in Plasma and Cerebrospinal Fluid as Potential Markers for Alzheimer’s Disease Takehiro Kikoa , Kiyotaka Nakagawaa,∗ , Tsuyoshi Tsudukib , Katsutoshi Furukawac , Hiroyuki Araic and Teruo Miyazawaa a Food
& Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan b Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan c Department of Geriatrics and Gerontology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
Accepted 6 September 2013
Abstract. The development of Alzheimer’s disease (AD) biomarkers remains an unmet challenge, and new approaches that can improve current AD biomarker strategies are needed. Recent reports suggested that microRNA (miRNA) profiling of biological fluids has emerged as a diagnostic tool for several pathologic conditions. In this study, we measured six candidate miRNAs (miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a) in plasma and cerebrospinal fluid (CSF) of AD and normal subjects by using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to evaluate their potential usability as AD biomarkers. The qRT-PCR results showed that plasma miR-34a and miR-146a levels, and CSF miR-34a, miR-125b, and miR-146a levels in AD patients were significantly lower than in control subjects. On the other hand, CSF miR-29a and miR-29b levels were significantly higher than in control subjects. Our results provide a possibility that miRNAs detected in plasma and CSF can serve as biomarkers for AD. Keywords: Alzheimer’s disease, cerebrospinal fluid, miRNA, plasma
INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia and a chronic neurodegenerative disease of the brain [1, 2]. For detecting and monitoring AD, in addition to minimally invasive tests, there are strategies for developing AD biomarkers by using metabolomics and proteomics techniques. However, identification of such biomarkers remains an unmet challenge [3–6], ∗ Correspondence
to: Kiyotaka Nakagawa, PhD, Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan. Tel.: +81 22 717 8904; Fax: +81 22 717 8905; E-mail: [email protected].
and new approaches that can improve current AD biomarker strategies are needed. An ideal biomarker should be specific, predictive, and easy to measure in an accessible fluid (e.g., blood and cerebrospinal fluid (CSF)). MicroRNAs (miRNAs) are a family of short noncoding RNAs whose final product is a 22-nucleotide functional RNA molecule that regulates the expression of target genes by binding to complementary regions of transcripts to repress their translation or cause mRNA degradation [7]. Aberrant expression of miRNAs has been implicated in diseases including cancer, viral hepatitis, and heart disease [8–11]. In addition to these diseases, since some miRNAs (e.g., miR-9, miR-29a,
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miR-29b, miR-34a, miR-125b, and miR-146a) have been reported to be expressed in mammalian brain [12], their importance in central nervous system (CNS) function and disease is now being recognized [13–15]. For example, miR-29a and miR-29b may facilitate increases in both amyloid- protein precursor (APP) and -site APP-cleaving enzyme (BACE1) in AD brain [16]. Other studies reported that oxidative stress and DNA damage, generated in part by elevations in amyloid- peptide (A), iron, and reactive oxygen species, would induce expression of miR-34a and miR146a [17, 18]. Based on these studies and together with recent reports about the occurrence of miRNAs in body fluids [19–23], the above candidate miRNAs in plasma and CSF of AD and normal subjects were analyzed by using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in order to evaluate their potential usability as AD biomarkers. To the best of our knowledge, there are only two reports of miRNA analysis in body fluids (CSF and white blood cells) of AD patients [24, 25], and the utility of plasma and CSF miRNAs as AD biomarkers for developing diagnostic tools and/or for monitoring pathology remains unclear. MATERIALS AND METHODS Ethics statement AD patient volunteers seen at the Tohoku University Hospital and healthy volunteer control subjects participated in this study (Table 1). Diagnosis of AD was set according to the NINCDS-ADRDA criteria [26]. Magnetic resonance imaging was taken in all the subjects. All the AD cases showed significant cerebral atrophy and no cerebrovascular lesions. Disease stage was rated by the Mini-Mental State Examination (MMSE), which is a brief cognitive test used widely in clinical practice and epidemiologic studies. This test was administered in order to grade the subjects’ global cognitive impairment. The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the Graduate School of Medicine at Tohoku University. All subjects gave written informed consent to the experimental protocol. Plasma and CSF samples from AD and healthy subjects Blood was collected into a tube containing EDTA2Na as an anticoagulant and was subjected to
Table 1 Physical characteristics of AD patients and control subjects1 Total number of subjects Males Females Age (years) MMSE CSF-A (1-42) (pg/ml) CSF-tau (pg/ml) 1 Means ± SD;
AD patients
Control subjects
10 3 7 80.7 ± 5.8 21.1 ± 3.5 378.4 ± 112.9 480.7 ± 218.9
10 4 6 73.0 ± 5.2 29.5 ± 0.7 560.4 ± 90.5 209.8 ± 64.2
n = 10.
centrifugation at 1,000 g for 10 min at 4◦ C. CSF samples were centrifuged at 500 g for 10 min at room temperature. Plasma and CSF were stored at −80◦ C until further analyzed. RNA isolation from plasma and CSF Total RNA, including miRNAs, was isolated from plasma or CSF with the miRNeasy kit (Qiagen). In brief, 2800 L of QIAzol reagent was added to 800 L of plasma or CSF samples. The sample was mixed with 560 L of chloroform in a tube. After mixing vigorously for 15 s, the sample was centrifuged at 12,000 g for 15 min. The upper aqueous phase was carefully transferred to a new collection tube, and 1.5 volume of ethanol was added. The sample was then applied directly to a silica membrane containing column and the RNA was bound and cleaned by using buffers provided by the manufacturer to remove impurities. The immobilized RNA was collected from the membrane with a low salt elution buffer. The RNA concentration was determined by a NanoDrop ND-1000 Spectrophotometers (Thermo Fisher Scientific Inc.). RNA quality was evaluated by an Agilent 2100 Bioanalyzer (Agilent Technologies) using an Agilent Small RNA kit. Real-time quantitative RT-PCR analysis Using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems), RNA was reverse transcribed to cDNA in the presence of 50 units of MultiScribe Reverse Transcriptase in a total volume of 15 L with the following conditions: 30 min at 16◦ C, 30 min at 42◦ C, and 5 min at 85◦ C. For qRT-PCR, TaqMan MicroRNA Assays (Applied Biosystems), including undiluted cDNA, 20×TaqMan MicroRNA Assay mix, TaqMan 2 × Universal PCR Master Mix, and nuclease-free water, were used. Each PCR reaction was performed in triplicate with MicroAmp™ optical 384-well reaction plates using a 7900HT Fast
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Fig. 1. miRNA levels in plasma of healthy human volunteers and AD patients. Values are mean ± SD (n = 10). Difference is considered significant from control subjects (p < 0.05).
Real-Time PCR System (Applied Biosystems), with reactions incubated at 95◦ C for 10 min, followed by 40 cycles of 95◦ C for 15 s and 60◦ C for 1 min. Fluorescence readings were taken during the 60◦ C step. The relative amounts of miRNAs were calculated by using the comparative cycle threshold (Ct) method. Human U6 and miR-24 served as an internal control and were used to normalize the plasma and CSF samples, respectively. CSF tau analysis CSF total tau was measured in duplicate using a double-sandwich enzyme-linked immunosorbent assay method (Innogenetics, Gent, Belgium) according to the manufacturer’s instructions. CSF Aβ analysis CSF A [1-42] was measured in duplicate using a double-sandwich enzyme-linked immunosorbent assay method (WAKO, Osaka, Japan) according to the manufacturer’s instructions.
RESULTS Plasma miRNAs in AD patients and healthy subjects The 6 miRNAs (miR-9, miR-29a, miR-29b, miR34a, miR-125b, and miR-146a) could be detected in plasma by qRT-PCR (Fig. 1). Plasma miR-34a and miR-146a levels in AD patients were significantly lower than in control subjects. The same tendency, although not significant, was observed for plasma miR-125b. No significant differences in plasma miR9, miR-29a, and miR-29b were found between AD patients and control subjects. CSF miRNA in AD patients and healthy subjects Similar to plasma, the six miRNAs could be detected in CSF by qRT-PCR (Fig. 2), and CSF miR-34a, miR125b, and miR-146a in AD patients were significantly lower than in control subjects. On the other hand, CSF miR-29a and miR-29b were significantly higher than in control subjects. No significant difference in CSF miR9 was found between AD patients and control subjects.
Statistical analyses Data are presented as mean ± SD. Differences in miRNA concentrations between control and AD subjects were compared using Student’s t-test or Welch’s t-test for equal or unequal variances; the MannWhitney U test was used when the distribution was skewed. A difference was considered significant at p < 0.05 or p < 0.01.
Relationship between miRNA and other parameters The levels of A and tau in the CSF of AD cases were lower and higher, respectively, than the mean of controls (Table 1). However, probably, the number of volunteers was small, and therefore, no significant correlation between the miRNAs levels in plasma or CSF
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Fig. 2. miRNA levels in CSF of healthy human volunteers and AD patients. Values are mean ± SD (n = 10). Difference is considered significant from control subjects (p < 0.05).
and other parameters (such as age, MMSE, CSF A, or CSF tau) of AD patients and control subjects was found (Supplementary Figs. 1–8). DISCUSSION miRNAs are recognized as key molecules in intracellular regulatory networks for gene expression, and the spectra and levels of some miRNAs are emerging as biomarkers for various pathological conditions [27]. Recent findings suggest that circulating miRNAs in blood plasma may be useful as biomarkers for the diagnosis of cancers [19, 21, 28, 29] and tissue injuries [30, 31]. In the present study, we selected miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a, because these 6 miRNAs were expressed in human brain and there were many reports about association of these miRNAs to AD mechanisms [32–34]. We measured the candidate 6 miRNAs (miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a) in plasma as well as CSF by qRT-PCR assays, and showed changes in circulating miRNA levels as a result of AD (Figs. 1, 2). It has been reported that in brains of AD patients, miR-29a and miR-29b are decreased, which leads to increased brain BACE1 expression [16]. BACE1 is the rate-limiting enzyme responsible for A production in the brain, and increased BACE1 expression is thought to be a risk for sporadic AD [35–37]. Additionally, the miR-29 family is reported to regulate DNA methyltransferase 3A and 3B [38]. Abnormal DNA methylation is usually found in AD brains and this can cause cell death [39]. Thus, decreasing miR-29a and miR-29b in brain would lead to increased A genera-
tion and DNA methylation, thereby becoming a burden for aged neurons [40]. In contrast to AD brain, in this study, we found that miR-29a and miR-29b were increased in CSF of AD patients (Fig. 2). Therefore, we might expect that miR-29a and miR-29b found in CSF originated from the brain, and these miRNAs were secreted from the AD brain to CSF. On the other hand, plasma miR-29a and miR-29b levels were unchanged (Fig. 1). Thus, it is hypothesized that miR-29a and miR-29b may be actively released from brain to CSF, but not to blood plasma. We need to clarify the mechanism of how these miR-29a and miR-29b levels were changed in brain and CSF, but not in plasma. miR-34a levels were reportedly increased in AD brain [41]. It has been shown that miR-34a overexpression can induce apoptosis in SH-SY5Y cells [42] utilizing the mechanism of interaction of miR-34a with the 3 -UTR of bcl-2 [42]. Bcl-2 is an antiapoptotic protein that prevents caspase-9 activation through an interaction with Apaf-1. Various studies have shown that bcl-2 overexpression is neuroprotective against apoptotic cell death or reduces the pathological changes in AD by inhibiting the activation of caspases [43, 44]. These effects of bcl-2 expression could be diminished by ectopic expression of miR-34a or knockdown of miR-34a [42]. In the present study, miR-34a was found to be decreased in both CSF and plasma of AD patients (Figs. 1, 2). Therefore, it was thought that miR-34a could not be secreted from AD brain into CSF and plasma. It is tempting to speculate that this may be one reason for AD development. However these are our hypotheses and further study is needed.
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Transcription of miR-146a is induced by pathophysiological stress factors, such as the pro-inflammatory cytokine IL-1 [45]. Relatively high levels of miR146a in brains of AD animal models as well as AD superior temporal lobe neocortex and hippocampus underscore the potential importance of miR-146a in neurodegenerative disease [45]. Upregulation of miR146a contributes to mediate innate immune response, inflammatory and synaptic pathobiology, and the manipulation of physiological stressors and environmental factors [45], which drive the AD process. In this study, miR-146a was found to be decreased in plasma and CSF of AD patients (Figs. 1, 2). Therefore, like miR-34a, miR-146a might accumulate in AD brain and not be released from brain to CSF and plasma. This may be one of the reasons for AD; however, research is needed to further understand the roles that these miRNAs play in AD. miR-9 and miR-125b are brain-enriched miRNAs [46, 47]. miR-9 has been associated with neurogenesis, morphogenesis, developmental patterning, brain cell proliferation, and glioblastoma [48, 49]. miR-9 and miR-125b increases in abundance in AD neocortex, suggesting that these brain-enriched miRNAs play some regulatory role in the degenerative process [47]. These miRNAs abundances are significantly changed during development and aging and exhibit a specifically altered expression profile in different regions of AD brain [50, 51]. In this study, no significant differences in plasma and CSF miR-9 were found between AD patients and control subjects (Figs. 1, 2). On the other hand, CSF miR-125b in AD patients was significantly lower than in control subjects, and the same tendency was observed for plasma miR-125b (Figs. 1, 2). As mentioned above, since both miR-9 and miR-125b are enriched in brain, the present findings may imply that these miRNAs are released from the brain in different ways. Further research is needed to evaluate the release of these miRNAs into the body fluids. Extracellular miRNAs circulating in the peripheral blood are included in cell membrane-derived particles, such as apoptotic bodies, microvesicles, and exosomes [52]. There is evidence that packaging of miRNAs into these particles results in protection from endogenous ribonucleases in plasma/serum [19]. For miRNAs in CSF, they showed a remarkable stability against exposure to exogenous RNase [53]. However, the role of extracellular miRNAs remains largely unknown, and additional studies exploring a potential biological function of miRNAs circulating in body fluids such as peripheral blood and CSF are required.
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Sample size of our study was rather small, and this may cause restriction to develop AD biomarkers. However, degree of data variation was satisfactory, and no outlying observations were found (Figs. 1 and 2), suggesting reliability of our data. This means that our present results could provide the rationale for future investigations of miRNAs in the plasma and CSF for diagnostic and prognostic purposes in a variety of CNS disease, including AD. To further clarify the usability for the biomarker of miRNAs, we need to show the results from large sample size. Such study will be presented in the near future as a different investigation. In conclusion, our results provide a possibility that miRNA detected in the CSF and plasma can serve as markers for AD. This finding is a preliminary indication of the abundant possibilities of using miRNAs to detect specific pathological conditions. In the present study of AD group, since we did not include other neurodegenerative disorders such as dementia with Lewy bodies and frontotemporal lobar degeneration, we consider that these miRNAs would be changed as a result of AD. For the possibility whether these miRNA change or not in other neurodegenerative diseases, this needs to examine in future experiments. There are no significant correlation between miRNAs and MMSE, CSF A, or CSF tau in both AD and control subjects. The reason may relate to plasma and CSF miRNA properties (incorporation into recipient cells followed by degradation, or urinary excretion), however these were still unclear and further study is needed. A more comprehensive study in humans is needed to determine the specificity and sensitivity of selected miRNA species. By means of qRT-PCR as well as miRNA array technologies, plasma and CSF-based miRNA markers that are specific for particular CNS disorders may be discovered in the future. Although the number of patients and controls studied here is small, our study provides the rationale for future investigations of miRNAs in the plasma and CSF for diagnostic and prognostic purposes in a variety of CNS disease, including AD. DISCLOSURE STATEMENT Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1944). SUPPLEMENTARY MATERIAL Supplementary tables are available in the electronic version of this article: http://dx.doi.org/10.3233/JAD130932.
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Journal of Alzheimer’s Disease 39 (2014) 253–259 DOI 10.3233/JAD-130932 IOS Press
MicroRNAs in Plasma and Cerebrospinal Fluid as Potential Markers for Alzheimer’s Disease Takehiro Kikoa , Kiyotaka Nakagawaa,∗ , Tsuyoshi Tsudukib , Katsutoshi Furukawac , Hiroyuki Araic and Teruo Miyazawaa a Food
& Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan b Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan c Department of Geriatrics and Gerontology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
Accepted 6 September 2013
Abstract. The development of Alzheimer’s disease (AD) biomarkers remains an unmet challenge, and new approaches that can improve current AD biomarker strategies are needed. Recent reports suggested that microRNA (miRNA) profiling of biological fluids has emerged as a diagnostic tool for several pathologic conditions. In this study, we measured six candidate miRNAs (miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a) in plasma and cerebrospinal fluid (CSF) of AD and normal subjects by using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to evaluate their potential usability as AD biomarkers. The qRT-PCR results showed that plasma miR-34a and miR-146a levels, and CSF miR-34a, miR-125b, and miR-146a levels in AD patients were significantly lower than in control subjects. On the other hand, CSF miR-29a and miR-29b levels were significantly higher than in control subjects. Our results provide a possibility that miRNAs detected in plasma and CSF can serve as biomarkers for AD. Keywords: Alzheimer’s disease, cerebrospinal fluid, miRNA, plasma
INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia and a chronic neurodegenerative disease of the brain [1, 2]. For detecting and monitoring AD, in addition to minimally invasive tests, there are strategies for developing AD biomarkers by using metabolomics and proteomics techniques. However, identification of such biomarkers remains an unmet challenge [3–6], ∗ Correspondence
to: Kiyotaka Nakagawa, PhD, Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan. Tel.: +81 22 717 8904; Fax: +81 22 717 8905; E-mail: [email protected].
and new approaches that can improve current AD biomarker strategies are needed. An ideal biomarker should be specific, predictive, and easy to measure in an accessible fluid (e.g., blood and cerebrospinal fluid (CSF)). MicroRNAs (miRNAs) are a family of short noncoding RNAs whose final product is a 22-nucleotide functional RNA molecule that regulates the expression of target genes by binding to complementary regions of transcripts to repress their translation or cause mRNA degradation [7]. Aberrant expression of miRNAs has been implicated in diseases including cancer, viral hepatitis, and heart disease [8–11]. In addition to these diseases, since some miRNAs (e.g., miR-9, miR-29a,
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miR-29b, miR-34a, miR-125b, and miR-146a) have been reported to be expressed in mammalian brain [12], their importance in central nervous system (CNS) function and disease is now being recognized [13–15]. For example, miR-29a and miR-29b may facilitate increases in both amyloid- protein precursor (APP) and -site APP-cleaving enzyme (BACE1) in AD brain [16]. Other studies reported that oxidative stress and DNA damage, generated in part by elevations in amyloid- peptide (A), iron, and reactive oxygen species, would induce expression of miR-34a and miR146a [17, 18]. Based on these studies and together with recent reports about the occurrence of miRNAs in body fluids [19–23], the above candidate miRNAs in plasma and CSF of AD and normal subjects were analyzed by using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in order to evaluate their potential usability as AD biomarkers. To the best of our knowledge, there are only two reports of miRNA analysis in body fluids (CSF and white blood cells) of AD patients [24, 25], and the utility of plasma and CSF miRNAs as AD biomarkers for developing diagnostic tools and/or for monitoring pathology remains unclear. MATERIALS AND METHODS Ethics statement AD patient volunteers seen at the Tohoku University Hospital and healthy volunteer control subjects participated in this study (Table 1). Diagnosis of AD was set according to the NINCDS-ADRDA criteria [26]. Magnetic resonance imaging was taken in all the subjects. All the AD cases showed significant cerebral atrophy and no cerebrovascular lesions. Disease stage was rated by the Mini-Mental State Examination (MMSE), which is a brief cognitive test used widely in clinical practice and epidemiologic studies. This test was administered in order to grade the subjects’ global cognitive impairment. The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the Graduate School of Medicine at Tohoku University. All subjects gave written informed consent to the experimental protocol. Plasma and CSF samples from AD and healthy subjects Blood was collected into a tube containing EDTA2Na as an anticoagulant and was subjected to
Table 1 Physical characteristics of AD patients and control subjects1 Total number of subjects Males Females Age (years) MMSE CSF-A (1-42) (pg/ml) CSF-tau (pg/ml) 1 Means ± SD;
AD patients
Control subjects
10 3 7 80.7 ± 5.8 21.1 ± 3.5 378.4 ± 112.9 480.7 ± 218.9
10 4 6 73.0 ± 5.2 29.5 ± 0.7 560.4 ± 90.5 209.8 ± 64.2
n = 10.
centrifugation at 1,000 g for 10 min at 4◦ C. CSF samples were centrifuged at 500 g for 10 min at room temperature. Plasma and CSF were stored at −80◦ C until further analyzed. RNA isolation from plasma and CSF Total RNA, including miRNAs, was isolated from plasma or CSF with the miRNeasy kit (Qiagen). In brief, 2800 L of QIAzol reagent was added to 800 L of plasma or CSF samples. The sample was mixed with 560 L of chloroform in a tube. After mixing vigorously for 15 s, the sample was centrifuged at 12,000 g for 15 min. The upper aqueous phase was carefully transferred to a new collection tube, and 1.5 volume of ethanol was added. The sample was then applied directly to a silica membrane containing column and the RNA was bound and cleaned by using buffers provided by the manufacturer to remove impurities. The immobilized RNA was collected from the membrane with a low salt elution buffer. The RNA concentration was determined by a NanoDrop ND-1000 Spectrophotometers (Thermo Fisher Scientific Inc.). RNA quality was evaluated by an Agilent 2100 Bioanalyzer (Agilent Technologies) using an Agilent Small RNA kit. Real-time quantitative RT-PCR analysis Using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems), RNA was reverse transcribed to cDNA in the presence of 50 units of MultiScribe Reverse Transcriptase in a total volume of 15 L with the following conditions: 30 min at 16◦ C, 30 min at 42◦ C, and 5 min at 85◦ C. For qRT-PCR, TaqMan MicroRNA Assays (Applied Biosystems), including undiluted cDNA, 20×TaqMan MicroRNA Assay mix, TaqMan 2 × Universal PCR Master Mix, and nuclease-free water, were used. Each PCR reaction was performed in triplicate with MicroAmp™ optical 384-well reaction plates using a 7900HT Fast
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Fig. 1. miRNA levels in plasma of healthy human volunteers and AD patients. Values are mean ± SD (n = 10). Difference is considered significant from control subjects (p < 0.05).
Real-Time PCR System (Applied Biosystems), with reactions incubated at 95◦ C for 10 min, followed by 40 cycles of 95◦ C for 15 s and 60◦ C for 1 min. Fluorescence readings were taken during the 60◦ C step. The relative amounts of miRNAs were calculated by using the comparative cycle threshold (Ct) method. Human U6 and miR-24 served as an internal control and were used to normalize the plasma and CSF samples, respectively. CSF tau analysis CSF total tau was measured in duplicate using a double-sandwich enzyme-linked immunosorbent assay method (Innogenetics, Gent, Belgium) according to the manufacturer’s instructions. CSF Aβ analysis CSF A [1-42] was measured in duplicate using a double-sandwich enzyme-linked immunosorbent assay method (WAKO, Osaka, Japan) according to the manufacturer’s instructions.
RESULTS Plasma miRNAs in AD patients and healthy subjects The 6 miRNAs (miR-9, miR-29a, miR-29b, miR34a, miR-125b, and miR-146a) could be detected in plasma by qRT-PCR (Fig. 1). Plasma miR-34a and miR-146a levels in AD patients were significantly lower than in control subjects. The same tendency, although not significant, was observed for plasma miR-125b. No significant differences in plasma miR9, miR-29a, and miR-29b were found between AD patients and control subjects. CSF miRNA in AD patients and healthy subjects Similar to plasma, the six miRNAs could be detected in CSF by qRT-PCR (Fig. 2), and CSF miR-34a, miR125b, and miR-146a in AD patients were significantly lower than in control subjects. On the other hand, CSF miR-29a and miR-29b were significantly higher than in control subjects. No significant difference in CSF miR9 was found between AD patients and control subjects.
Statistical analyses Data are presented as mean ± SD. Differences in miRNA concentrations between control and AD subjects were compared using Student’s t-test or Welch’s t-test for equal or unequal variances; the MannWhitney U test was used when the distribution was skewed. A difference was considered significant at p < 0.05 or p < 0.01.
Relationship between miRNA and other parameters The levels of A and tau in the CSF of AD cases were lower and higher, respectively, than the mean of controls (Table 1). However, probably, the number of volunteers was small, and therefore, no significant correlation between the miRNAs levels in plasma or CSF
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Fig. 2. miRNA levels in CSF of healthy human volunteers and AD patients. Values are mean ± SD (n = 10). Difference is considered significant from control subjects (p < 0.05).
and other parameters (such as age, MMSE, CSF A, or CSF tau) of AD patients and control subjects was found (Supplementary Figs. 1–8). DISCUSSION miRNAs are recognized as key molecules in intracellular regulatory networks for gene expression, and the spectra and levels of some miRNAs are emerging as biomarkers for various pathological conditions [27]. Recent findings suggest that circulating miRNAs in blood plasma may be useful as biomarkers for the diagnosis of cancers [19, 21, 28, 29] and tissue injuries [30, 31]. In the present study, we selected miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a, because these 6 miRNAs were expressed in human brain and there were many reports about association of these miRNAs to AD mechanisms [32–34]. We measured the candidate 6 miRNAs (miR-9, miR-29a, miR-29b, miR-34a, miR-125b, and miR-146a) in plasma as well as CSF by qRT-PCR assays, and showed changes in circulating miRNA levels as a result of AD (Figs. 1, 2). It has been reported that in brains of AD patients, miR-29a and miR-29b are decreased, which leads to increased brain BACE1 expression [16]. BACE1 is the rate-limiting enzyme responsible for A production in the brain, and increased BACE1 expression is thought to be a risk for sporadic AD [35–37]. Additionally, the miR-29 family is reported to regulate DNA methyltransferase 3A and 3B [38]. Abnormal DNA methylation is usually found in AD brains and this can cause cell death [39]. Thus, decreasing miR-29a and miR-29b in brain would lead to increased A genera-
tion and DNA methylation, thereby becoming a burden for aged neurons [40]. In contrast to AD brain, in this study, we found that miR-29a and miR-29b were increased in CSF of AD patients (Fig. 2). Therefore, we might expect that miR-29a and miR-29b found in CSF originated from the brain, and these miRNAs were secreted from the AD brain to CSF. On the other hand, plasma miR-29a and miR-29b levels were unchanged (Fig. 1). Thus, it is hypothesized that miR-29a and miR-29b may be actively released from brain to CSF, but not to blood plasma. We need to clarify the mechanism of how these miR-29a and miR-29b levels were changed in brain and CSF, but not in plasma. miR-34a levels were reportedly increased in AD brain [41]. It has been shown that miR-34a overexpression can induce apoptosis in SH-SY5Y cells [42] utilizing the mechanism of interaction of miR-34a with the 3 -UTR of bcl-2 [42]. Bcl-2 is an antiapoptotic protein that prevents caspase-9 activation through an interaction with Apaf-1. Various studies have shown that bcl-2 overexpression is neuroprotective against apoptotic cell death or reduces the pathological changes in AD by inhibiting the activation of caspases [43, 44]. These effects of bcl-2 expression could be diminished by ectopic expression of miR-34a or knockdown of miR-34a [42]. In the present study, miR-34a was found to be decreased in both CSF and plasma of AD patients (Figs. 1, 2). Therefore, it was thought that miR-34a could not be secreted from AD brain into CSF and plasma. It is tempting to speculate that this may be one reason for AD development. However these are our hypotheses and further study is needed.
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Transcription of miR-146a is induced by pathophysiological stress factors, such as the pro-inflammatory cytokine IL-1 [45]. Relatively high levels of miR146a in brains of AD animal models as well as AD superior temporal lobe neocortex and hippocampus underscore the potential importance of miR-146a in neurodegenerative disease [45]. Upregulation of miR146a contributes to mediate innate immune response, inflammatory and synaptic pathobiology, and the manipulation of physiological stressors and environmental factors [45], which drive the AD process. In this study, miR-146a was found to be decreased in plasma and CSF of AD patients (Figs. 1, 2). Therefore, like miR-34a, miR-146a might accumulate in AD brain and not be released from brain to CSF and plasma. This may be one of the reasons for AD; however, research is needed to further understand the roles that these miRNAs play in AD. miR-9 and miR-125b are brain-enriched miRNAs [46, 47]. miR-9 has been associated with neurogenesis, morphogenesis, developmental patterning, brain cell proliferation, and glioblastoma [48, 49]. miR-9 and miR-125b increases in abundance in AD neocortex, suggesting that these brain-enriched miRNAs play some regulatory role in the degenerative process [47]. These miRNAs abundances are significantly changed during development and aging and exhibit a specifically altered expression profile in different regions of AD brain [50, 51]. In this study, no significant differences in plasma and CSF miR-9 were found between AD patients and control subjects (Figs. 1, 2). On the other hand, CSF miR-125b in AD patients was significantly lower than in control subjects, and the same tendency was observed for plasma miR-125b (Figs. 1, 2). As mentioned above, since both miR-9 and miR-125b are enriched in brain, the present findings may imply that these miRNAs are released from the brain in different ways. Further research is needed to evaluate the release of these miRNAs into the body fluids. Extracellular miRNAs circulating in the peripheral blood are included in cell membrane-derived particles, such as apoptotic bodies, microvesicles, and exosomes [52]. There is evidence that packaging of miRNAs into these particles results in protection from endogenous ribonucleases in plasma/serum [19]. For miRNAs in CSF, they showed a remarkable stability against exposure to exogenous RNase [53]. However, the role of extracellular miRNAs remains largely unknown, and additional studies exploring a potential biological function of miRNAs circulating in body fluids such as peripheral blood and CSF are required.
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Sample size of our study was rather small, and this may cause restriction to develop AD biomarkers. However, degree of data variation was satisfactory, and no outlying observations were found (Figs. 1 and 2), suggesting reliability of our data. This means that our present results could provide the rationale for future investigations of miRNAs in the plasma and CSF for diagnostic and prognostic purposes in a variety of CNS disease, including AD. To further clarify the usability for the biomarker of miRNAs, we need to show the results from large sample size. Such study will be presented in the near future as a different investigation. In conclusion, our results provide a possibility that miRNA detected in the CSF and plasma can serve as markers for AD. This finding is a preliminary indication of the abundant possibilities of using miRNAs to detect specific pathological conditions. In the present study of AD group, since we did not include other neurodegenerative disorders such as dementia with Lewy bodies and frontotemporal lobar degeneration, we consider that these miRNAs would be changed as a result of AD. For the possibility whether these miRNA change or not in other neurodegenerative diseases, this needs to examine in future experiments. There are no significant correlation between miRNAs and MMSE, CSF A, or CSF tau in both AD and control subjects. The reason may relate to plasma and CSF miRNA properties (incorporation into recipient cells followed by degradation, or urinary excretion), however these were still unclear and further study is needed. A more comprehensive study in humans is needed to determine the specificity and sensitivity of selected miRNA species. By means of qRT-PCR as well as miRNA array technologies, plasma and CSF-based miRNA markers that are specific for particular CNS disorders may be discovered in the future. Although the number of patients and controls studied here is small, our study provides the rationale for future investigations of miRNAs in the plasma and CSF for diagnostic and prognostic purposes in a variety of CNS disease, including AD. DISCLOSURE STATEMENT Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1944). SUPPLEMENTARY MATERIAL Supplementary tables are available in the electronic version of this article: http://dx.doi.org/10.3233/JAD130932.
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