MicroRNA super-resolution imaging in blood for Alzheimer’s disease

Abstract

We propose a novel blood biomarker detection method that uses miRNA super-resolution imaging to enable the early diagnosis of Alzheimer’s disease (AD). Here, we report a single-molecule detection method for visualizing disease-specific miRNA in tissue from an AD mice model, and peripheral blood mononuclear cells (PBMCs) from AD patients. Using optimized Magnified Analysis of Proteome (MAPs), we confirmed that five miRNAs contribute to neurodegenerative disease in the brain hippocampi of 5XFAD and wild-type mice. We also assessed PBMCs isolated from the whole blood of AD patients and a healthy control group, and subsequently analyzed those samples using miRNA super-resolution imaging. We detected more miR-200a-3p expression in the cornu ammonis 1 and dentate gyrus regions of 3 month-old 5XFAD mice than in wild-type mice. Additionally, miRNA super-resolution imaging of blood provides AD diagnosis platform for studying miRNA regulation inside cells at the single molecule level. Our results present a potential liquid biopsy method that could improve the diagnosis of early stage AD and other diseases.

INTRODUCTION

Alzheimer’s disease (AD), a neurodegenerative disease, is the most common characterized by the presence of amyloid beta (Aβ) plaques and hyperphosphorylation of tau protein in the brain (1). An ideal diagnostic technique for AD should be minimally invasive, low-cost, easily applied in mass screenings, and able to identify the disease with adequate reliability before symptoms appear for which treatment is the most effective. The current diagnostic techniques for AD are cognitive testing (2), structural imaging with magnetic resonance imaging (MRI) or computed tomography (3), and biomarker detection (4). However, MRI is an expensive and specialized technique that is logistically unsuitable for mass screening. The mini-mental state exam (MMSE) (5), which is widely used to evaluate the cognitive function of AD patients, can return a normal finding at a disease stage in which behavioral disorders do not appear, and therefore cannot predict or provide an early diagnosis for the disease. Recently, the development of a positron emission tomography (PET) technique that can photograph β-amyloid accumulation in the brain, known as the cause of AD, has opened the way for early AD diagnosis. However, PET tests are expensive, which limits their broad application (6).

Another access is to screen for biomarkers in the peripheral blood (7). Specific changes in blood-based RNA profiles, such as whole peripheral blood and blood fractionations, may be able to reflect physiological and pathological processes that occur in various cells and tissues throughout the body (8). Consistently, peripheral blood cells were found to share more than 80% of the transcriptome in major tissues including the brain, heart, kidney, liver, and spleen (9). Therefore, detecting biomarkers in peripheral blood presents a path more suitable for mass screenings and the regular monitoring of disease progression than current methods (10).

MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that plays an important role in the post-transcriptional regulation of gene expression through binding to the 3’-untranslated regions of target messenger-RNAs (mRNAs) (11). Many studies have demonstrated the expression of specific proteins and miRNAs in neurodegenerative diseases and the central nervous system (12). For example, lower expression of miR-195 has been shown in AD-patients (2), and has-miR-151a-3p and has-miR-261a-5p are differentially expressed in the peripheral blood mononuclear cells (PBMCs) from Parkinson’s disease (PD) patients and controls (13). Also, both hsa-miR-107 and hsa-miR-103 were downregulated in the blood of AD, PD, major depression, and schizophrenia patients, and upregulated in patients with mild cognitive impairment (MCI), multiple sclerosis, and bipolar disorder (14). Therefore, miRNA–disease associations can broaden our understanding of the molecular mechanisms of various human diseases and provide new prognostic biomarkers.

Ku et al. developed the magnified analysis of the proteome (MAP) protocol that provides 4-fold expansion of the cellular architecture and shows three-dimensional proteome organization while protecting endogenous proteins in hydrogel form (15). We modified the original MAP techniques (Optimized Cell MAP and Paper-MAP) to enable high-resolution imaging analyses that preserve fluorescent signals and intracellular cellular structures through a 4-fold expansion of biological samples, such as single cells, organoids, and brain tissues, such as brain tumor (16, 17). Due to technical limitations, accurately imaging endogenous or unmanipulated RNA in cells without false positive signals was previously impossible. In this study, we propose a miRNA super-resolution imaging method that can detect miRNAs in blood-derived cells for early disease diagnosis.

Our novel technique for miRNA quantification and visualization in PBMCs and tissues does not require gene amplification. We identified significant expression of miR-200a-3p in tissue from an AD mouse model, and verified that those results correspond with miRNA quantified from serum by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Our MAP-based miRNA super-resolution imaging method in blood is convenient, inexpensive, accurate, and fast, making it an exciting potential clinical approach for early disease diagnosis. Rapid AD-specific miRNA liquid biopsy in blood using our MAP-based miRNA super-resolution imaging technique could be a non-invasive diagnostic tool for AD in the future.

RESULTS

miR-200a-3p significantly expressed in cornu ammonis 1 of 5XFAD mouse brains

To investigate the difference in miRNA expression between WT and 5XFAD hippocampus-tissues by super-resolution imaging analysis, we first generated the 4-fold expanded and transparent hydrogel tissues in hippocampus tissue via Paper-MAP. Fig. 1A shows that 200 μm thick hippocampal tissue sections of 5XFAD and wild-type (WT) mice were 4-fold expanded via Paper-MAP technique containing tissue clearing, denaturation, and expansion processes. We also applied a MAP-based novel tool for miRNA quantification and three-dimensional visualization in PBMCs without a required gene amplification (Fig. 1B). In our quantification analysis, we selected 1) miRNAs that tended to increase with the progression of Alzheimer’s disease (AD), and 2) miRNAs that were expected to be secreted extracellularly, and were thus potential biomarkers in serum.

Fig. 1.

Schematic of MAP process with tissue and blood. Schematic representation of PBMC isolation from patient blood, the Paper-MAP process for mouse brains (A), and liquid biopsy for blood (B).

Using a conventional confocal laser microscope, we performed 3D and volumetric imaging of nuclear patterns after immunostaining for SYTO17 (red fluorescent nucleic acid stain) to visualize miRNA expression of quantum dots (QD)–miRNA molecular beacon (MB) in the hippocampus samples of WT and 5XFAD mice. We sought differentially expressed miRNA in cornu ammonis (CA1, CA2, and CA3) and dentate gyrus (DG) hippocampal areas of the hippocampus tissue processed via Paper-MAP. We found a highly significant increase in the expression of miR-200a-3p, especially in the CA1 and DG hippocampal areas of 3 month-old 5XFAD mice (Fig. 2). However, 9-month-old WT and 5XFAD mice showed decreased expression of miR-200a-3p, which reflects aging and an advanced disease stage. We thus confirmed that miR-200a-3p increases early in the progression of AD.

Fig. 2.

Differential miRNA expression profiles in the hippocampal-regions of wild-type and 5XFAD mice via Paper-MAP. miR-200a-3p, miR-7-5p, miR-671-5p, and SYTO-17 immunostaining in the hippocampi of mouse brains processed via Paper-MAP. Each image (white box: CA1, CA2, CA3, DG) was taken with the same 40× objective lens and z-stacked for comparison. Green arrows indicate miR-200a-3p expression. Scale bars: yellow = 1000-μm, blue = 30-μm.

Next, we also found that the expression of miR-7-5p and miR-671-5p in early stage 3 month-old 5XFAD mice did not differ significantly from that of the age-matched WT mice (see at the bottom of the Fig. 2). miR-7-5p expression increased in the CA2 region at 9 month-old WT, compared with the CA2 region at 3 month-old WT (Supplementary Fig. 1 of the Supplementary Information [SI]). Many recent studies have shown that miR-7 is involved in brain development process and the pathological processes of various brain diseases (18). miR-671-5p is not expressed in AD animal model, but functions as a tumor suppressor in various cancers (19). ciRS-7-5p, which acts as a potent miR-7 sponge, and miR-122-5p were not expressed in mice aged (3-9) months, despite increased exposure intensity (Supplementary Fig. 2 of the SI). We thus found that particular miRNAs, such as miR-671-5p, ciRS-7-5p, and miR-122-5p, are consistently deregulated throughout AD progression. A previous study that found molecular mechanisms of ciRS-7 depletion in AD suggested that ciRS-7 could be an AD treatment target (20). Similarly, we present results for lesser-known miRNA circulating in AD tissue samples. Our findings indicate that the progressive pathology of AD can cause miRNA changes.

Imaging and qRT−PCR analyses of miRNA expression in mice

Next, we examined the expression of miR-200a-3p in different mouse-tissues and quantified miRNA spot images in the hippocampal regions using a 3D rendering tool (Fig. 3A, B; and Supplementary Fig. 3 of the SI). To identify a potentially novel serum biomarker that could be used to identify or monitor AD pathogenesis in the brain, qRT-PCR was used to confirm miR-200a-3p expression (Fig. 3C). We identified differentially expressed miR-200a-3p among 4 representative groups: early and late stage at (1-3) and (6-9) months AD, respectively, in 5XFAD mice and age-matched early and late stage WT mice.

Fig. 3.

miRNA expression in wild-type and 5XFAD brain hippocampus via Paper-MAP. (A) SYTO-17 immunostaining in hippocampus-sections of mouse brains processed via Paper-MAP. Each image (white box: CA1, CA2, CA3, DG) was taken with the same 40× objective lens and z-stacked for comparison. Green arrows indicate miR-200a-3p expression. Scale bars: yellow = 100-μm. (B) Spot counting of miR-200a-3p (QD525) in wild-type (3 months; n = 6, 9 months; n = 6) and 5XFAD mouse hippocampal-regions at early-stage AD (3 months, n = 6) and late-stage AD (9 months, n = 6), processed via 3D-Rendering. (C) qRT-PCR quantification of miR-200a-3p expression in serum from 5XFAD and wild-type mice at early-stage AD and late-stage AD. Means ± SD from triplicate experiments are shown. *P < 0.05, ^†P < 0.01.

In the early stage groups, we found that miR-200a-3p expression in the hippocampal-tissue and blood was significantly higher in the 5XFAD mice than in the WT mice. In other words, upregulation of miR-200a-3p expression in serum from disease model mice correlated with the increase in miRNA in the CA1 and DG of hippocampal tissue. After the advancement of AD (at the late stage), the miRNA expression in 5XFAD mice was decreased, compared to the early stage. Thus, these analyses confirm that specific miRNA is dynamically expressed across AD relative to the WT. These results show that the variations observed in miRNA levels are not simply a result of variation in preanalytical processing, but do indeed reflect disease progression. Our work therefore significantly expands our understanding of mouse hippocampal miRNAs, and is useful for obtaining accurate results in specific hippocampal areas of the brain.

We also examined whether lectins and blood vessel structure differed between the 5XFAD and WT mice (Supplementary Fig. 4A of the SI). The 3D images show decreased blood vessel volume and increased length in the 5XFAD group, compared with the WT group. Furthermore, we found a dense distribution of amyloid beta (Aβ) neuroimaging biomarkers between the blood vessels in the hippocampus during AD progression (Supplementary Fig. 4B of the SI). In the 5XFAD group, the hippocampus, which is essential for memory and cognitive function, contained extracellular Aβ plaques, a distinct pathological hallmark of AD, confirming the presence of both amyloid pathology and hippocampal atrophy due to neuronal cell death (21). Therefore, Aβ can be proposed as a potential biomarker that could be used to monitor disease progression.

miRNA super-resolution imaging and qRT−PCR analysis in human PBMCs

To investigate changes in a neurodegenerative marker during AD progression, we identified differentially expressed miR-200a-3p among 3 representative patient groups: the control group with normal cognitive function (NC), the group with mild cognitive impairment (MCI), and the group with dementia (AD) (student’s t-test, P < 0.05) (Fig. 4). In this study, we considered data from 28 patients with a diagnosis of MCI or AD, pathological levels of amyloid confirmed by PET (POS. or N/A), and MMSE score ≥ 20. Using our technique and the MB probes that can detect fluorescence only when hybridized to a target sequence, we observed the presence of miRNAs. The expression of miR-200a-3p was higher in the nuclei of PBMCs from AD patients than in those from the NC group, and those results were quantified using spot counting (Fig. 4A, B). It is difficult to accurately diagnose MCI (the stage between preclinical disease and the clinical development of dementia) in animal models. However, our results confirmed a significant increase in miR-200a-3p expression in PBMCs from patients diagnosed with MCI (Fig. 4B). We also evaluated miR-200a-3p in the serum of AD patients using a TaqMan Advanced miRNA array (Fig. 4C), and found that miR-200a-3p expression was higher in the AD group than in the MCI group.

Fig. 4.

miRNA expression in Alzheimer’s disease patients. (A) Representative images of miR-200a-3p in serum from the cognitively normal control (NC), mild-cognitive-impairment (MCI), and Alzheimer’s-disease (AD)-groups. (B) Spot counting of miR-200a-3p (QD525 or QD565) in PBMCs from AD-patients (n = 8) compared with the NC (n = 5) group, processed via 3D-Rendering. (C) miR-200a-3p expression levels in AD-patients versus the NC-group by qRT-PCR. Validation by qRT-PCR in patients with AD (n = 20) and NC (n = 5) confirmed that miR-200a-3p showed significantly higher serum expression in AD-patients than in controls. Means ± SD are shown from triplicate-experiments. *P < 0.05, ^†P < 0.01.

In addition, we observed significant differences in the expression levels when we expanded our analysis to four groups [Control, MCI, AD, and early-onset AD (EOAD)] (Supplementary Fig. 5 of the SI). In EOAD, the expression of miR-200a-3p was increased 5-fold compared with the MCI-group, and increased (2 to 3)-fold compared with the AD group. Samples of rare diseases, such as EOAD, are difficult to obtain from patients, and difficult to reproduce in animal models. However, if miRNA super-resolution imaging for blood can confirm that the expression of AD-related miRNAs increases before proteins, such as Aβ, Tau, and amyloid precursor protein (APP), accumulate in the brain, EOAD could be diagnosed quickly. Taken together, our results show that changes in miRNA expression are associated with the progression of AD.

DISCUSSION

Alzheimer’s disease (AD) is characterized by extracellular accumulation of amyloid beta (Aβ) plaque (22) and intracellular neurofibrillary tangles of hyperphosphorylated tau proteins (23). AD is a slowly progressing disease (24), and various miRNAs have been suggested as potential diagnostic or prognostic biomarkers (25). Early detection of such miRNA changes through a liquid biopsy could enable the early treatment of AD. In this study, we modified the liquid biopsy technique to analyze miRNAs. miRNAs play important roles in various physiological and pathological processes by mediating the expression of target mRNAs. miRNAs are also released from cells, and enter into circulatory biological fluids including blood plasma/serum, saliva, and urine (26). Interestingly, Liang et al. (14) showed that the brain and blood PBMCs expression patterns cluster together, which might indicate that a specific blood-based expression signature could be used as a biomarker for AD and other neurological diseases. Previous studies have introduced several candidate biomarkers in the blood and blood cells, but their sensitivity, specificity, and true association with brain processes remain unclear (27). Another barrier to current methods is that it is not possible to accurately determine whether the miRNA in serum (identified by qRT-PCR) and the miRNA in PBMCs (identified by miRNA super-resolution imaging in blood) have the same origin. In addition, the identification of reliable blood biomarkers for AD diagnosis continues to be limited by technical issues (28). Most studies are based on qRT-PCR and the TaqMan detection system for detecting RNA (e.g., miRNA) that is present in low concentrations in the blood. However, the above methods are not adequately accurate, and inaccurately detected miRNA levels in experiments can greatly affect the diagnostic utility of molecules. Therefore, in-depth small molecule RNA analyses are needed to sensitively investigate small quantitative changes in miRNA expression.

We previously observed intracellular miRNA by developing an optimized Cell–MAP technique that can preserve fluorescence while maintaining cellular structures (16). Specifically, we reported the 3D structures of miR-122 and miR-671 in Hep-3B cells. We also showed that Paper-MAP enables the visualization of miRNAs in expanded tissues. In this study, we attempted to further determine whether miRNAs present in blood, as reported in previous studies, could be identified from tissues prepared using Paper-MAP.

One of the keys to research investigating miRNAs as biomarkers for AD is the verification of such biomarkers in diverse patient cohorts. Nagaraj et al. (29). reported finding hsa-miR-200a-3p in plasma separated from mild cognitive impairment (MCI) patients diagnosed with probable early AD. In another study, increased levels of hsa-miR-200a-3p were reported in blood mononuclear cells from early AD patients (12), and Lau et al. (30) investigated increases in miR-200a-3p in the brain cortex. Similarly, our imaging results verified the presence of hsa-miR-200a-3p in PBMCs isolated from the blood of MCI and AD patients, with a 6-fold increase in MCI, and a 7-fold increase in AD, compared with the NC group. These results suggest that this miRNA can be secreted by blood mononuclear cells. However, because miRNAs are detected in or around the nucleus, both the precursor form and mature form are probably reflected in our results. It is currently impossible to accurately distinguish the precursor from the mature form, so further studies using a precursor beacon system are needed. Our qRT-PCR results show a (3 to 4)-fold increase in the serum levels of hsa-miR-200a-3p in AD patients, compared with the NC group. Of note, this study represents the first time in which miRNA expression is profiled in liquid biopsies (e.g., blood sample), with two optimized MAP methods and super-resolution imaging analysis. Our results also indicate that miRNA imaging from liquid biopsies can detect MCI faster than qRT-PCR. Furthermore, our results are similar to previous studies showing increased miR-200a-3p expression in the brain of AD patients and in the hippocampus of AD mouse model (29-31). Studies indicated that hsa-miR-200a-3p plays a critical role in the biological processes such as the neuroinflammation, inflammatory response, and apoptosis by modulating expression of several target mRNAs such as SIRT1 (32). SIRT1 is an anti-apoptotic protein that has a protective role in several neurodegenerative diseases such as Alzheimer’s, Parkinson’s and motor neuron diseases (27). Several studies have described the function of hsa-miR-200a-3p, but the exact molecular mechanisms of Alzheimer’s disease progression are still unknown. Therefore, further studies on the miR-200a-3p target gene pathway following AD progression are needed. Compared to conventional methods, this technique offers new opportunities to advance current understanding of disease pathogenesis, drug discovery, medical diagnoses, and biological research.

In addition, the MB technology used in this methodology can be used to detect a variety of DNA and RNA targets through its unique on/off signal mechanism. Therefore, the fluorescence intensity is proportional to the number of specific miRNAs in a sample, making the technique highly sensitive and specific. Accurately imaging endogenous or unmanipulated RNA in cells without false positive signals was previously impossible. Utilizing MAP-based super-resolution imaging and MB technology, we propose a miRNA super-resolution imaging method that can detect miRNAs in blood-derived cells for early disease diagnosis. This is the first study to visualize miRNAs present in the blood and determine accurate amounts of miRNAs without non-specific reactions.

In conclusion, our MAP-based miRNA super-resolution imaging method can provide simple, reproducible, and rapid miRNA liquid biopsy analyses. However, this process has not yet been standardized, and further improvements are needed to produce the most efficient method. For example, the gel components should be modified to improve gel solidity, so that each sample can be reused. Further validation of use in liquid biopsies and extension to other human biofluids are also needed.

MATERIALS AND METHODS

Materials and methods are available in the supplemental material.

Funding Statement

ACKNOWLEDGEMENTS This study was supported by a research grant from Gangnam Severance Hospital, Yonsei University College of Medicine. This study was supported by a faculty research grant from the Yonsei University College of Medicine for (6-2020-0109). This work was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (grant number: HI17C1260 and HU20C0164). This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1F1A1072307). This work was supported by the Basic Science Research Program through the NRF, funded by the Ministry of Education (NRF-2020R1A6A3A01097969).