Elevated levels of MicroRNA-455-3p in the cerebrospinal fluid of Alzheimer’s patients: a potential biomarker for Alzheimer's disease

Alzheimer’s disease (AD) is a progressive, degenerative disorder manifested by dementia in aged individuals (1). Over 50 million people worldwide are currently living with AD-related dementia, and this number is expected to increase to 152 million by 2050 (World Alzheimer Report 2019). AD-related dementia has huge economic consequences, as indicated by the total worldwide healthcare annual cost of dementia estimated in 2019 at $1 trillion (2). In addition to dementia, AD is associated with the loss of synapses, synaptic dysfunction, mitochondrial structural and functional abnormalities, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aβ), and phosphorylated-tau (p-tau) (3–6). Despite the progress that has been made in better understanding AD pathogenesis, researchers have still not identified early detectable biomarkers, and drugs/agents that can prevent AD or slow its progression. Recent studies showed that microRNAs (miRNAs) could be ideal diagnostic and therapeutic candidates due to their previous roles in AD (7–9). Neuronal and synaptic miRNAs are involved in multiple synaptic functions such as synaptic plasticity, synaptogenesis, synaptic scaling, excitability, and synaptic integrity that are crucial for neurotransmission and cognitive function (10).

MicroRNA-455–3p (miR-455–3p) is identified as a member of the broadly conserved miRNA family expressed in most of the phylum and species. The role of miR-455 has been implicated in various human diseases such as cartilage development, adipogenesis, preeclampsia, and cancers, e.g., colon cancer, prostate cancer, hepatocellular carcinoma, renal cancer, oral squamous cancer, skin cancer, and non-small cell lung cancer (11). Recently, our laboratory discovered the biomarker and therapeutic relevance of miR-455–3p in AD (12).

The goal of our present study is to identify the potential peripheral circulatory miRNAs as the biomarkers for AD. Our research journey on miR-455–3p was started with global microarray analysis of serum samples from AD patients, mild cognitive impaired (MCI) individuals, and healthy subjects, which showed the high level of miR-455–3p in AD patients relative to MCI and healthy controls (12). Further, validation analysis using different kinds of AD samples such as a serum, AD postmortem brains, AD fibroblasts, AD B-lymphocytes, cell lines treated with Aβ, and AD mouse model (Tg4576) confirmed the high level of miR-455–3p in AD and could be a potential biomarker (12,13). We also determined the mechanistic link of miR-455–3p in AD via modulation of mutant APP and Aβ levels (14). The high level of miR-455–3p reduces Aβ toxicity, enhances mitochondrial biogenesis and synaptic activity, and maintains healthy mitochondrial dynamics (14). Luciferase reporter assay confirmed the APP as a validated target of miR-455–3p. Studies on mouse neuroblastoma cells revealed the protective role of miR-455–3p against Aβ-induced toxicities. We also noticed that miR-455–3p enhances cell survival and lifespan extension (14).

We studied the status of miR-455–3p in almost all kinds of AD sources to ensure its biomarker potential for AD. However, our study missing the data on miR-455–3p status in the cerebrospinal fluid (CSF) samples of AD patients and healthy control subjects. MiR-455–3p levels in the CSF is very important to determine the brain-specific location and secretion of miR-455–3p in AD. Therefore, the current study is dedicated to determining the brain-specific localization and secretion of miR-455–3p in AD CSF.

This work is a continuation of our ongoing big project ‘miR-455–3p and AD’. As mentioned previously, we identified a high level of miR-455–3p in different kinds of AD samples. The miR-455–3p is validated on all these sources and confirmed the significant relevance of miR-455–3p with AD. Since AD is a brain disorder and miR-455–3p level is higher in AD postmortem brains, so it is important to determine the status of miR-455–3p in AD CSF. Therefore, in this study, we determined the status of miR-455–3p in AD CSF.

CSF samples from AD patients and unaffected healthy controls were obtained from NIH NeuroBioBank center- Human Brain and Spinal Fluid Resource Center, 11301 Wilshire Blvd (127A), Los Angeles, CA. Thirty-one CSF samples including AD patients (n=20) and age and sex-matched unaffected healthy controls (n=11) were received from NIH NeuroBioBank. Demographic details of the samples provided in Supplementary Table 1 and neuropathology reports of the AD patients were summarized in Supplementary Table 2. The study was conducted at the Internal Medicine Department of Texas Tech University Health Sciences Center, and the study protocol was approved by the Institutional Review Board of TTUHSC, Lubbock, Texas for the use of biospecimens in Project FRONTIER (IRB #: L06–028).

Exosomes were extracted from the CSF samples of AD patients and unaffected healthy controls by using Total Exosome Isolation (from other body fluids) kit (Invitrogen, Catalog Number: 4484453). Briefly, 500 μl of CSF samples were mixed with an equal amount of extraction reagent and mixed by vortexing until the solution is homogenous. The resulting homogenate was incubated at 4°C for 1 hour. After incubation, samples were centrifuged at 10,000×g for 1 hour. The supernatant was discarded and the pellet containing the exosomes was processed for exosome characterization and RNA extraction.

The exosome extract was characterized by transmission electron microscopy (TEM) and immunoblotting analysis of exosome markers. The quality of exosome preparation from the CSF was determined by TEM analysis. Exosome pellet was washed with 1X PBS and dissolved in a fixative solution (8% glutaraldehyde, 16% paraformaldehyde, and 0.2M sodium cacodylate buffer) for 1 h at room temperature. Samples were centrifuged at 300 g for 3 min and the resulting cell pellet was undergone electron microscopy at the Imaging Core Facility at Texas Tech University. TEM images showed the 100 nm-distinct structures of exosome vesicles (Fig. 1A). Further, exosomes were characterized by immunoblotting analysis of exosome markers CD6 and CD63 (15). Exosomal proteins were extracted from AD and controls samples and immunoblotted with CD9 and CD63 antibodies by using ExoAb Antibody Kit (System Biosciences, CA, USA). The levels of CD9 and CD63 proteins are detected in both AD and unaffected healthy control exosomes (Fig. 1B).

Figure 1-.

(A) Representative TEM images of exosomes extracted from CSF of AD patients (100 nm magnification). (B) Immunoblotting of exosomal markers CD9 and CD63 in AD patients and unaffected healthy controls. (C) Relative expression of miR-455–3p in the CSF exosomes of AD patients and healthy controls. (D) ROC curve for miR-455–3p level in AD patients’ vs healthy controls. (E) MiR-455–3p level correlation with amyloid plaque counts in AD patient’s brain.

After CSF exosomes characterization, the total RNA was isolated from the exosome pellet by using the TriZol RT reagent (Ambion, USA) as per manufacturer instructions. The expression of miR-455–3p was quantified by qRT-PCR. Quantification involved 3 steps: (i) Polyadenylation. One μg of total RNA was polyadenylated with a miRNA First-Strand cDNA synthesis kit (Agilent Technologies Inc., CA, USA), following the manufacturer’s instructions. (ii) cDNA synthesis. Ten μL of polyadenylated miRNAs were processed for cDNA synthesis with the miRNA First-Strand cDNA synthesis kit (Agilent Technologies Inc., CA, USA). (iii) Real-time qRT-PCR. Real-time qRT-PCR was performed by preparing a reaction mixture containing 1 μL of miRNA-specific forward primer (10μm), 1 μL of a universal reverse primer (3.125 μM) (Agilent Technologies Inc., CA, USA), 10 μL of 2X SYBR Green PCR master mix (Applied Biosystems CA), and 1 μL of cDNA. To this mixture, RNase-free water was added to a total of 20-μL final volume. Primers used in the current study were synthesized commercially (Integrated DNA Technologies, Inc., City, Iowa, USA) for miR-455–3p and U6 small nuclear RNA (snRNA). To normalize the miRNA expression U6 snRNA expression was also quantified in the same samples, which was used as an internal control. The reaction mixture for each sample prepared in triplicates was set in the 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). MiRNA fold changes were calculated by using the formula (2^−ΔΔct) (13–15).

The real-time qRT-PCR data were compared between the unaffected healthy controls and AD patients. The analysis showed the high expression of miR-455–3p in the CSF exosomes of AD patients compared to controls (P=0.0164) (Fig. 1C). Further, to evaluate the diagnostic value of miR-455–3p in AD CSF, we performed the receiver operating characteristic (ROC) curve analysis of miR-455–3p in AD samples relative to unaffected healthy controls. ROC curve analysis showed the significant AUROC value for miR-455–3p (AUROC=0.745; P=0.0232) (Fig. 1D). This data indicates that miR-455–3p levels increased in the brain during AD progression and drives the release of miR-455–3p from the brain to CSF. These observations again confirmed the diagnostic capabilities of miR-455–3p in AD. We also did the correction analysis of miR-455–3p levels with the number of amyloid plaques per 200X of neuropathology reports of ten AD samples, since we do not have the amyloid plaques counts for all samples (Supplementary Table 2). MiR-455–3p showed a positive correlation with the number of amyloid plaques (R=0.576), though it was not significant (Fig. 1D).

In summary, the piece of work presented here is a continuation of our ongoing research miR-455–3p project. For the first time, we discovered the high level of miR-455–3p in AD serum and further validation of miR-455–3p in different AD sources confirmed a significant biomarker relevance of miR-455–3p with AD. However, miR-455–3p biomarker validation is incomplete until it is determined in the CSF of AD patients. Therefore, in the current study, we determined the exosome secretion from the brain that contains a high level of miR-455–3p. This finding adds up a piece of additional evidence to support that miR-455–3p could be a peripheral biomarker for AD assessment. Currently, we are extensively investigating the cellular and molecular functions of miR-455–3p in AD using miR-455–3p transgenic and miR-455–3p knockout mouse models. However, future research is warranted to determine the neuronal localization of miR-455–3p and in context to synaptic function.