MicroRNA Profile in Patients with Alzheimer’s Disease: Analysis of miR-9-5p and miR-598 in Raw and Exosome Enriched Cerebrospinal Fluid Samples

Abstract

Background: MicroRNAs have been postulated as potential biomarkers for Alzheimer’s disease (AD). Exosomes are nanovesicles which transport microRNAs, proteins, and other cargos. It has been hypothesized that the exosome traffic might be increased in neurodegenerative disorders.

Objective: i) To assess the cerebrospinal fluid (CSF) microRNA profile in a group of AD patients and control subjects and to validate a group of microRNAs previously reported by other authors. ii) To compare microRNA levels in whole CSF and in the exosome-enriched fraction in AD patients.

Methods: A panel of 760 microRNAs was analyzed in the CSF of 10 AD patients and 10 healthy subjects. Among microRNAs differently expressed, we selected those that had been previously reported by other authors. Candidates were validated in a larger group by individual qPCR assays. MicroRNA expression was also evaluated in exosome-enriched CSF samples of patients with AD and controls.

Results: Fifteen microRNAs were differently expressed in AD. MiR-9-5p, miR-134, and miR-598 were selected as candidates for further analysis. MiR-9-5p and miR-598 were detected in 50 and 75% of control CSF samples, respectively, while they were not detected in any AD CSF samples. We observed an opposite pattern when we evaluated the microRNA expression in the exosome-enriched CSF AD samples. No pattern variations were noted among healthy subjects.

Conclusion: These data propose miR-9-5p and miR-598 as potential biomarkers for AD. Further studies in plasma and other body fluids will confirm their potential role as easily accessible biomarkers. In addition, our data suggest that exosome trafficking is different between AD and control subjects raising the need to take this phenomenon into consideration in future studies of AD biomarkers.

Keywords

Alzheimer’s disease biomarkers exosomes microRNAs

INTRODUCTION

Alzheimer’s disease (AD) is the most frequent neurodegenerative dementia [1], characterized by progressive memory impairment and deterioration of other cognitive domains such as orientation, language, and behavior [2]. Histopathologically, the AD brain is characterized by the deposition of both neuritic plaques composed of amyloid-β (Aβ) peptides and hyperphosphorylated forms of the microtubule-associated protein tau in neurofibrillary tangles [3]. AD biomarkers can be divided into: cerebrospinal fluid (CSF) biomarkers (Aβ, total-tau, and phosphorylated-tau), and MRI and PET biomarkers [4, 5].

MicroRNAs have recently emerged as a potential biomarker tool for different diseases, such as cancer and inflammatory disorders [6]. MicroRNAs are endogenous noncoding ribonucleic acids, usually 21–25 nucleotides long, that modulate gene expression at the post-transcriptional level [7]. MicroRNAs inhibit the translation of mRNA and/or promote their degradation by binding to the 3’ untranslated region (3’-UTR) [8]. Each microRNA targets hundreds of genes while each individual gene is targeted by multiple microRNAs. They are transcribed in the nucleus and afterwards they are exported to the cytoplasm. Once there, they undergo a maturation process to become functionally active. They can act intracellularly or be secreted into exosomes and transported to other cells or tissues [9]. MicroRNAs have been suggested as biomarkers in neurodegenerative diseases. Of note, in AD, some authors have identified different microRNAs in CSF, serum, and plasma samples [10 –20]. Some of those microRNAs are involved in different cellular functions such as neurogenesis, cell proliferation, immune response, or microglial-mediated neuronal injury [15]. However, the lack of reproducible results across studies still question the role of microRNAs as potential biomarkers for AD [21, 22].

enlargethispage *2ptExosomes are small membrane vesicles (size ranging between 30 and 100 nm) that have attracted interest in recent years. These vesicles have been isolated in body fluids, such as blood, urine, saliva, and CSF. Exosomes are involved in critical biological functions, such as immune response, antigen presentation, intracellular communication, and the transfer of RNA and proteins [23]. As shown in the ExoCarta exosome database, a wide variety of molecules are present in exosomes, including proteins, lipids, DNA, mRNAs, and microRNAs [24]. Several studies have demonstrated an increased exosomal response in some neurodegenerative processes, such as Parkinson’s disease, amyotrophic lateral sclerosis, and AD [25 –27]. In addition, altered microRNA profiles have been recently reported in CSF exosomes in both Parkinson’s disease and AD [28]. However, how the interactions between microRNA and exosome dynamics take place and their potential roles in AD remain unclear [25, 29].

Although several groups have explored the patterns of microRNA expression in different biofluids in AD, their role has not been elucidated yet. In this study, we evaluated the microRNA profile in CSF from patients with AD and compared it with a group of healthy subjects in order to assess its potential role as a biomarker for the disease. In addition, we assessed whether the microRNA traffic through exosomes is increased in AD. To do so, we analyzed the presence of microRNAs in whole CSF samples and exosome-enriched samples of patients with AD and control subjects.

MATERIAL AND METHODS

Patients, CSF data, and study design

Patients were part of a cohort which had been previously studied with clinical and neuropsychological assessment, as well as by determining their AD biomarkers profile (core biomarkers in CSF [Aβ, total-tau, and phosphorylated-tau]) [30]. All patients or their relatives had previously given written consent for CSF extraction and for inclusion into the investigation cohort. In this study, all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki and carried out according to the international Good Laboratory Practice (GLP) and Good Clinical Practice (GCP) standards. The study protocol was approved by the Institutional Review Board.

Two groups of subjects were studied: i) healthy controls with no memory impairments and with negative AD biomarkers; and ii) patients with a clinical presentation consistent with AD, and positive CSF AD biomarkers. Cohort data are shown in Table 1.

In the present study, we first analyzed the whole microRNA profile in 10 individual CSF samples of AD patients and 10 individual CSF samples of control subjects. This discovery set served to select the potential candidate microRNAs. Afterwards, we confirmed the results of the selected candidates in a wider sample of 18 cases and 18 controls (main data summarized in Table 1) by single real time quantitative polymerase chain reaction (rt-qPCR) assays. In addition, to assess the microRNAs traffic through exosomes in AD, we also evaluated whether the levels of the candidate microRNAs differed when they were extracted from CSF exosome-enrichedsamples.

CSF collection and AD biomarkers analysis

CSF samples were acquired by lumbar puncture following the recommendations of Lewczuk et al. [31]. All CSF samples were free of blood contamination. After collection, CSF samples were centrifuged (1600 g, 4°C, 15 min), aliquoted, and stored at – 80°C until used.

CSF biomarkers were measured in our laboratory using commercial ELISA kits (Innotest-Aβ₄₂ [Art. no. 81576]; Innotest t-tau [Art. no. 81572]; Innotest p-tau [Art. no. 81574]) following the manufacturer’s instructions. Cut-off values for AD were established in our laboratory: <663.5 pg/ml for Aβ₄₂, >456 pg/ml for t-tau, and >68 pg for p-tau.

MicroRNA extraction and reverse transcription

CSF samples (300μL) were used for RNA isolation. For both microRNA extraction and reverse transcription (RT) we used Exiqon commercial kits, following the manufacturer’s instructions (Biofluids kit (PN 300112), Universal cDNA Synthesis kit (PN203301)).

In addition, we also isolated microRNAs from exosome-enriched CSF fractions, obtained by Exosome isolation kits (PN300102), Exiqon), following the manufacturer’s instructions.

Study of the microRNAome profile and rt-qPCR

For studying the microRNAome in the CSF samples, we used the Exiqon microRNA panel (http://www.exiqon.com/microRNA-pcr-panels; product number 203615) and followed the manufacturer’s instructions. This panel evaluates up to 752 human microRNAs. We used this platform to individually analyze 10 CSF samples from AD patients and 10 CSF samples from controls. Then, we identified candidate microRNAs that were differently expressed in AD patients. In a second step, we carried out an external confirmation, by selecting, among the differentially expressed microRNAs, those that had been previously reported in the literature as differentially expressed in AD patients.

Afterwards, we aimed to confirm the differential expression of those microRNAs by using rt-qPCR in a larger group of subjects (18 AD patients, 18 healthy controls). In this validation phase, we used individual PCR assays with specific probes for the candidate microRNAs, provided by Exiqon. The amplification reaction was monitored with SYBR Green dye included in a master mix (Exilent SYBR Green master mix (PN203421), Exiqon). The amplification reactions were carried out on an ABI 7300 thermal-cycler.

Exosome-enriched CSF samples and evaluation of the exosome-associated microRNA expression

For this purpose, we used aliquots of 20 samples (10 AD patients, 10 controls) that had been previously used for the microRNAs analysis in whole CSF. The exosome-enriched fraction of the CSF samples was obtained with the miRCURY Exosome Isolation Kit (https://www.exiqon.com; product number 300102) according to the manufacturer’s instructions. For standard microRNA extraction, we used 300μL aliquots of CSF. Briefly, the protocol consists of: i) thawing samples on ice; ii) pelleting of dead cells/debris; iii) mixing of the sample with proprietary precipitation buffer and overnight incubation; iv) pelleting of the exosomal fraction and finally; v) exosome lysis and standard microRNA extraction protocol (described above).

Data analyses

In the microRNAome evaluation, each microRNA Ct (cycle threshold) was normalized by the average Ct in the whole plate of that sample. Then, the relative expression of each microRNA was calculated as 2 - ^ΛCt, where ΔCt is the normalized Ct of each particular microRNA. Subsequently, the differences between the AD and control groups were computed as the fold-changes between the average expressions of both groups of subjects. The significance of the differences was tested by Mann Whitney U test.

Then, for the purposes of this study we considered as differentially expressed those microRNAs that fulfilled the following conditions: i) consistently detected (>60% of samples) in at least one of the groups; ii) between-group fold change differences >2; and iii) unadjusted p-value < 0.05.

For the selection of the housekeeping microRNAs we took advantage of the Normfinder software included in GenEx software (GenEx; http://www.multid.se/genex/genex.html. Thus, miR-22-3p and miR-124-3p, which were consistently detected in both groups, were chosen as normative microRNAs for individual rt-qPCR assays.

In the validation phase, all individual qPCR assays were done in triplicate. Samples showing poor reproducibility across replicates were repeated. Both AD and control samples were randomized and included in each experiment to diminish the influence of between-run variation. Likewise, negative non-template controls and UniSP6-spiked positive controls were included. For the qualitative analysis, a Ct of 36 was considered, so microRNAs with Ct below 36 were regarded as expressed. The frequency of expression in AD and control groups was then compared with the exact Fisher’s test.

RESULTS

MicroRNAoma panel in CSF

Globally, among the 752 microRNAs, up to 205 of them were detected at least in the 40% of AD CSF samples. This number increased up to 239 in the case of CSF control subject samples. Hence, overall microRNA levels tended to be somewhat lower in AD (Fig. 1A). By contrast, 346 and 381 microRNAs were not detected in any AD-CSF or Control-CSF samples, respectively (Raw data provided as Supplementary Material). Due to the limited sample availability and the low levels of microRNAs in CSF, we used the whole samples for the analysis and we did not specifically check the quality of microRNAs. Nevertheless, the average Cts were very similar in both groups of subjects (34.93 and 34.99 in the panel analyses for the more than 200 microRNAs expressed in AD and control groups, respectively), thus suggesting that input RNA and quality were similar in both groups.

After microRNAome analysis, 14 differentially-expressed microRNAs were identified: miR-877-5p, miR-206, miR-20a-5p, miR-9-5p, miR-491-5p, miR-320a, miR-126-5p, miR-150-5p, miR-509-3p, miR-26a-5p, miR-141-3p, miR-598, miR-188-5p, and miR-134 (Table 2). In addition, miR-22-3p and miR-124-3p were initially selected as housekeeping genes to normalize the results.

Afterwards, in order to externally validate our previous microRNA candidates, we compared our results with those previously reported in the literature by other groups [10 –12]. As a result, we eventually selected 3 candidates: i) miR-9-5p, ii) miR-134, and iii) miR-598 (Fig. 1B).

Validation by individual qPCR assays

To confirm our previous results, we used 18 AD and 18 control CSF samples. Each group was composed by the samples used for the microRNA panel analyses as well as 8 AD and 8 control additional CSF samples (Table 1). In this phase, in a wide proportion of samples, the microRNA candidates were not detected, or detected at very low concentrations by rt-qPCR. It is worth mentioning that in order to avoid background noise, we considered as truly expressed only those microRNAs detected at a threshold cycle <36. By using these criteria, none of the candidate or housekeeping microRNAs were detected in the AD group (mean Ct values obtained in this validation phase are summarized in Table 3). However, we found important differences among the candidate microRNAs. MiR-9-5p was detected in 50% of the control samples, while it was not detected in any AD CSF sample (Fisher exact test; p = 0.001). Regarding miR-598, it did not appear in any AD patient either, while it was consistently detected in almost 75% of controls (Fisher exact test; p < 0.001). No significant differences were found for miR-134 (that was detected in 2 control and none of the AD CSF samples)(Fig. 2A).

The role of exosomes in microRNA expression among AD patients and control subjects

In the light of some studies that postulated that intercellular communication through exosomes might be increased under some external and internal stimuli, including the neurodegeneration process, we explored whether the exosomal response was upregulated in AD. We measured the levels of our candidates (miR-9-5p, miR-134, and miR-598) in exosome-enriched purified CSF fractions of 10 AD and 10 control subjects. Then, we compared the levels of those microRNAs (measured by qPCR) in whole CSF and in the exosome-enriched fraction. Surprisingly, we observed a marked inversion in the expression pattern previously described. Indeed, a tendency of microRNA overexpression in AD patients was observed when microRNAs were analyzed in the exosome-enriched fraction (Fig. 2B). Thus, miR-9-5p and miR-598 were detected in 7 out 10 and up to the 80% of exosome-enriched CSF samples from AD, respectively (Fisher Test, p = 0.003 and p = 0.0007, respectively, when comparing with AD non-exosome enriched CSF sample). No significant differences between the raw and exosome-enriched fractions were found regarding miR-134. In contrast to AD, in control subjects we did not find significant differences in the microRNA detection rates between raw and exosome-enriched CSF samples (data not shown). In line with these results, there was a tendency of increased microRNA expression in the exosome-enriched fractions of samples with AD, in comparison with controls, but the difference did not reach statistical significance (Fig. 2C; Table 4).

DISCUSSION

The search of new and more accessible biomarkers for both stratifying and identifying subjects at risk to develop a specific disease is a pressing need in neurodegenerative diseases. In this line, microRNAs have been postulated as a useful biomarker tool for these diseases, including AD [8].

Several studies have evaluated microRNA expression in the CSF of AD patients. Interestingly, in keeping with our findings, all of them showed that there was a tendency toward microRNA underexpression in AD patients [10–14 , 21]. However, the results are poorly consistent across studies, with a clear lack of reproducibility [12 , 21]. In this scenario, our work was conceived to explore CSF microRNA expression in our set of AD patient samples as well as to try to validate some of the results previously described in the literature. Therefore, when we analyzed the microRNAome profile, we selected as candidates only those microRNAs that had been previously described as differentially-expressed by other groups. In this line, miR-9-5p had already been pointed out by both Kiko et al. and byBurgos et al., while miR-598 and miR-134 only by the later [11, 12]. MiR-9-5p had been reported as differently expressed in both CSF and serum samples of AD patients, while variations in miR-598 and miR-134 had only been described in CSF[11 , 33].

Since housekeeping microRNAs were only detected at very low levels by qPCR, in order to avoid “false positives”, we decided to do a qualitative analysis of the results. In this line, the detection of both miR-9-5p and miR-598 in CSF was significantly underrepresented in AD, supporting the concept that these microRNAs might be downregulated as a response to the neurodegenerative process. Interestingly, our results do agree with those published by Kiko et al. and Burgos et al., who described decreased expression of both miR-9-5p and miR-598 [11, 12].

A complementary in silico analysis by using the miRSystem ver. 20160513 webtool (http://mirsystem.cgm.ntu.edu.tw/) showed that these microRNAs were potential regulators of gene pathways related to the nervous system biology, such as amyloid proteins, stress pathways, and neurotrophic signaling, supporting a potential role in the pathogenesis of AD. Regarding other biofluids, namely serum, onlymiR-9-5p has been reported as differently expressed in AD. Since these fluids are much more easily collectable, further studies are warranted to assess the concentration of the microRNAs we have found in such fluids. Regarding exosomes, the experience with AD is still limited. Several studies have discussed their role in neurodegenerative disease, based upon some of their functions such as misfolded protein exportation, lipid or protein cargo, and mRNA and microRNA transport [25 , 29]. In AD, in line with the tendency observed in our study, an increased exosome traffic has been reported by several authors [34, 35]. In this context, Goetzl et al. described altered lysosomal proteins in neural-derived plasma exosomes in preclinical AD [34]. However, to the best of our knowledge, our study is the first one comparing microRNA levels in exosome-enriched CSF fractions with microRNAs in raw CSF samples. Our results might suggest that exosome trafficking is altered in AD. Nevertheless, further research is required to fully elucidate the precise role of exosomes in AD. In this line, microRNA expression and exportation seemed to be linked to these vesicles in patients with AD. Although further studies are needed to delineate the exact role of exosome trafficking in AD, our results have an important practical implication for the design of future studies, in relation with the discordant microRNA levels in unenriched and exosome-enriched CSF fractions.

Several limitations should be noted in this study. Firstly, the sample size is limited and, therefore, these results should be considered as exploratory, deserving further confirmation in other population groups. Secondly, microRNAs are at very low concentration in CSF. Hence, the methodology used in this study might not be sensitive enough to detect differences in microRNAs present at the lowest concentration range. Although a qPCR procedure was used for both the discovery and the validation phases, subtle differences between both assays might help to explain some of the differences found in the detection of some microRNAs at both stages.

In conclusion, in our study starting from a wide microRNAome panel we have identified two potential CSF candidates that had been previously reported by other authors in AD patients. Further studies in other body fluids such as plasma or urine will elucidate if these microRNAs could have a role as peripheral biomarkers. In addition, our data suggest that the CSF microRNA trafficking through exosomes is increased in patients with AD. In view of these findings, future studies evaluating the whole microRNAome expression in exosome-enriched CSF samples are needed in order to elucidate the exact nature of the exosomal changes in AD. Finally, our data suggest that studies addressing microRNA expression must take into consideration the potential influence of changes in exosome trafficking. This is particularly relevant if microRNA levels are to be considered as diagnostic or prognostic tools.

Footnotes

ACKNOWLEDGMENTS

This study was supported by CIBERNED (“SIGNAL Study” and “Mecanismos epigenéticos implicados en la etiología y progresión de las demencias rápidamente progresivas (PI2014/02) Study”.

Authors’ disclosures available online ().

Appendix

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