The plasma derived exosomal miRNA-483-5p/502-5p serve as potential MCI biomarkers in aging

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Introduction
The public's health is in peril due to the increasing incidence of dementia (Giusti et al., 2014;Aranda et al., 2021).The latest study found that 4253 of 49,202 individuals from Europe and North America developed dementia, the incidence rate reached to 8.6 % (Wolters et al., 2020).By 2050, dementia prevalence will nearly double in Europe and triple globally (Prince, 2015;Scheltens et al., 2021).A clinic trial showed lecanemab that recently received regulatory approval in the US and Japan, reduced markers of amyloid in early Alzheimer's disease and resulted in moderately less decline on measures of cognition (van Dyck et al., 2023).Donanemab, has also shown that it can slow the rate of progression of Alzheimer's disease in trial data published this.But none of the medicines are a cure for Alzheimer's Disease (Sims et al., 2023).Therefore, early detection of AD-related pathophysiological changes, especially during the Mild Cognitive Impairment (MCI) stage, remains a pivotal goal for effective intervention.MCI is usually described as an intermediate phase between normal cognition and dementia, where includes at least one area of measurable cognitive impairment (Voisin et al., 2003).The study demonstrated that the transient MCI can return to its normal state through proper intervention (Nussbaum and Ellis, 2003).Conservative estimates place the conversion rate from MCI to dementia at 5 %-10 % year (Roberts et al., 2014;Knopman et al., 2015).Therefore, early diagnosis to intervention of MCI may postpone the occurrence of dementia.A quarter of all dementia patients worldwide reside in China (Collaborators GBDN, 2019).A study found that in China, those over 65 years old had a dementia prevalence of 5.60 % (Huang et al., 2019).As a result, a large number of people will have dementia and cognitive impairment.The majority of research to date has been on the Southern Chinese inpatient population, leaving the occurrence of MCI in the underdeveloped Northwestern China uncertain.The study commenced by selecting a neighborhood in a northern city located in a less developed area as its starting point.Furthermore, there is a pressing requirement to enhance the detection methods by incorporating dependable, non-invasive biomarkers for MCI.While biomarkers like Abeta42, t-tau, p-tau in cerebrospinal fluid (CSF), plasma clusterin, p53, and inflammatory molecules such as lactate and cytokines have shown promise in identifying individuals with MCI at risk of progressing into Alzheimer's disease (AD), the practical hindrances lie in the cost and unstable performance of CSF, along with the unclear accuracy of blood biomarkers (Anderson, 2019).Exosomes, small membranous vesicles released by various cell types, have emerged as key players in intercellular communication, shuttling bioactive molecules, including proteins, lipids, and nucleic acids, between cells.Recent research has unveiled a promising avenue in the form of extracellular vesicles, particularly exosomes, and the microRNAs (miRNAs) they harbor.Their dysregulation has been implicated in various pathological conditions, including neurodegenerative diseases (Giau et al., 2019).Dan et al.'s research found that microRNA (miRNA) exhibits significant diagnostic efficacy in cognitive disorders, showing significant properties of improved sensitivity and specificity.This is particularly evident in diagnostic modalities that employ a panel of miRNA assays, thus emphasizing their potential utility in a comprehensive diagnostic framework (Shi et al., 2020).As exosome extraction and purification techniques improve, research is gradually shifting to exosomal miRNAs because lipid compounds can protect miRNAs from RNase degradation, which holds great promise for disease detection (de Jong et al., 2012).According to the research, miR-132 showed a high sensitivity and specificity in distinguishing MCI from age-matched controls and was connected with the Montreal Cognitive Assessment score in MCI patients (Sheinerman et al., 2012;Xie et al., 2015).MicroRNA-206, which is known to be raised in the brains of Alzheimer's disease (AD) patients and decreases the expression of brain-derived neurotrophic factor, showed a 7.8-fold increase in mild cognitive impairment subjects and was also correlated with the MCI Montreal Cognitive Assessment score (Xie et al., 2015;Moon et al., 2016).MiR-483-5p, the novel AD biomarker candidate, whose fold rise was exceedingly significant when comparing MCI-AD and AD patients to non-demented controls in earlier studies (8-13 fold change) (Nagaraj et al., 2017).The expression of miR-502-5p predicted prognosis in patients with neural system malignancies (Drusco et al., 2018).Based on the amassed evidence, we have selected miRNA-132-3p, − 206, − 483-5p, and 502-5p as prospective candidates.The objective of this investigation is to assess the cognitive well-being of senior adults in northwest China and explore the potential utility of non-invasive exosomal miR-NAs for the early detection of MCI.

Recruitment of participants and sample collection
We obtained written consent from all subjects.Recruitment and random selection of 275 participants with signed informed consent were done using a list of register numbers.This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Lanzhou University School of Basic Medicine (Lzujcyxy20230354).During the entire recruitment and investigation process, we require family members to accompany us, and pay close attention to the subjects' mood and physical status so that can be terminated early.We started surveying cognitive function in people over 65 years old from September 2019.And participants who develop any of the following symptoms will be terminated from participating in this recruitment study: are diagnosed with dementia or other neurodegenerative diseases; diseases that impair cognitive function; suffer from severe mental illness; and are unable to communicate normally, or unable to complete the study due to poor cooperation, hearing impairment, visual impairment, or any other disability.The questionnaire scale consists of four parts: demographic characteristics, lifestyle and habits, history of chronic diseases including hypertension, diabetes, coronary heart disease (CHD) and Montreal Cognitive Assessment (MoCA).The criteria for identification of MCI included: ① Cognitive decline complaints from the patients or their families, ② The reporting of a decline in cognitive functioning relative to the previous year by the patient or informant is integral to assessing cognitive decline.③ Cognitive disorders as evidenced by clinical evaluation (impairment in memory or in another cognitive domain, here, the clinical evaluation shows a score < 26 by MoCA, on the contrary, MoCA ≥26 is considered normal), ③ Absence of major repercussions on daily life (the patient may, however, report difficulties concerning complex day-to-day activities), ④ absence of dementia (There is no memory impairment as the main manifestation, affecting daily work and life.), as provided by the MCI Working Group of the European Consortium on Alzheimer's Disease, 2006 (Portet et al., 2006).As the biomarker exploration cohort, 24 MCI patients and 24 agematched controls were chosen at random.A control group supplemented with participants younger than 65 and in good health was enrolled from the same neighborhood.The exclusion criteria should be as follows: older than 65 years; with a history of illnesses or injuries that have an impact on cognitive function; any inflammation that is acute; people who are unable to converse.

Sample preparation
Whole blood samples were collected in EDTA coated tubes and centrifuged at 1500 xg for 15 min at 4 • C. The upper layer of plasma was transferred to a 15 ml sterile polypropylene tube on ice.After centrifugation again at 3200 xg for 15 min in 4 • C, platelets were removed.Plasma was then aliquoted into 1 ml per tube, snap frozen and stored at − 80 • C within 90 min of blood collection (Shi et al., 2010).

Exosome isolation and identification
Plasma protein quantification was measured by Bicinchoninic acid assay (BCA) kit and the concentration was presented in Table 3.For uniformity and quantification of exosome isolation, paired pool plasma containing 100 mg protein were prepared.Briefly, plasma was centrifuged at 2000 xg for 15 min at 4 • C, and 12,000 g for 30 min at 4 • C before ultracentrifugation (100,000 g, 3 h, 4 • C) for exosome collection.At the end of the second ultracentrifuge, the platelet was resuspended with 200 μl ice cold phosphate buffer (PBS) (Beyotime, C0221A).

Electron microscopy
The exosome mixture was applied to copper grid and negatively stained with 2 % phosphotungstic acid.Transmission image of exosome were acquired by transmission electron microscope (TEM) (FEI Tecnai G2 Spirit Bio-Twin).

miRNAs extraction and purification and real-time PCR
miRNAs in exosome were extracted using the miRcute miRNA Isolation Kit (TIANGEN, DP503) according to the manufacturer's instruction with a minor modification.The first strand and real-time PCR were conducted according to the manufacturer's instruction (TIANGEN: miRcute plus miRNA first-strand cDNA Kit, KR211-02; miRcute plus miRNA qPCR Kit (SYBR GREEN), FP411-02).An external control (TIANGEN, CD200-01) was utilized for miRNA normalization.Primers used were presented in Table 1.All results were analyzed using 2 •△△Ct and were shown as mean ± SEM.

Pathway enrichment analysis
The target genes of these four miRNAs (miRNA-132-3p, miR-206, miR-483-5p and miR-502-5p) were retrieved from three online databases: miRDB, TargetScan, and miRTarBase.The compiled target gene lists were subjected to pathway enrichment analysis using the Metascape online platform (http://metascape.org).Metascape integrates data from various sources, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathways.Default parameters were used for the analysis, and enriched terms with a P-value <0.05 were considered significant.The network analysis was performed to visualize the relationships between enriched terms.The resulting network was visualized using Cytoscape (Shannon et al., 2003).

Statistical analysis
Data analyzing were conducted by SPSS software version 26.0 and GraphPad Prism 8.0.Prevalence of MCI in people with 65 years old and over were analyzed by Chi-square test depending on different demographic characteristics.Comparison of exosome release and variance of exosomal miRNA expressions among groups were completed by oneway or two-way ANOVA analysis.The ROC analysis was performed to assess the diagnostic utility of selected miRNAs in discriminating between MCI.Receiver Operating Characteristic (ROC) curves were constructed using miRNA-132-3p, miR-206, miR-483-5p and miR-502-5p as potential discriminators.The Area Under the Curve (AUC) was calculated to quantify the discriminatory power of each miRNA.Sensitivity, specificity, and other relevant measures were derived to further evaluate the diagnostic performance.

The demographic and clinical characteristics of aging people from the perceptive of cognitive function
A total of 275 participants were enrolled, including 118 females (42.9 %) and 157 males (57.1 %), respectively.99 participants were found to have MCI based on the criteria of the European Alzheimer's Disease Consortium in 2006.The frequency rate was 36.0 %.Our investigation and analysis showed statistical significance in four aspects, including age (Pearson χ2 = 83.146，P< 0.001), educational level (Pearson χ2 = 52.196，P< 0.001), income (Pearson χ2 = 6.18，P = 0.045), keeping regular exercise (Pearson χ2 = 5.716，P = 0.017).The details are presented in Table 2.

miRNAs identified in enhanced pathways for head development, modulation of chemical synaptic transmission and tube morphogenesis
In an effort to derive biological meaning, we conducted a comprehensive analysis of the targeted genes of four microRNAs (miRNA-132-3p, miR-206, miR-483-5p and miR-502-5p) using three prominent online databases, namely miRDB, TargetScan, and miRTarBase.The enrichment analysis on these target genes indicates that the four microRNAs (miRNA-132-3p, miR-206, miR-483-5p and miR-502-5p) involved in regulating genes associated with tube morphogenesis, cellular responses to growth factors, modulation of chemical synaptic transmission and the development of structures within the head region (Fig. 1).The discovery of these regulatory networks adds to our understanding of the molecular mechanisms behind the biological effects of the investigated miRNAs, emphasizing their potential as therapeutic plasma derived exosomal biomarkers in MCI.

The cohort for miRNA detection as biomarker for MCI
The miRNA-study cohort included 24 MCI patients, 24 age-matched control and 24 supplemented control group below 65 years.Demographic information, MoCA score and concentration of plasma protein were presented in Table 3.

Plasma exosome identification
We first isolated and characterized plasma exosomes by WB and TEM.The purity of the exosome was confirmed by the presence of exosome associated proteins (Alix, CD9 and TSG101) (Fig. 2a).The morphology of the exosome by TEM indicated a membrane bound particle with a diameter around 100 nm (Fig. 2b).

Comparison of exosome release in control groups and MCI patients
As exosomes possess multiple roles in physical and pathological conditions, it was intriguing to compare the exosome release among the three groups.30 μg plasma protein were loaded onto SDS-PAGE and staining of Coomassie blue showed an equivalent content of protein between health control and MCI groups (Fig. 3a).CD9 showed an increasing trend dependent on age.TSG101 in the group of HC>65y and MCI were also higher than that in HC<65y.There was no certain pattern of variation in Alix, but MCI showed less than that in normal control (Fig. 3a).We didn't find apparent significant differences of these three markers among the groups (Fig. 3b-d).

Discussion
This study investigated the cognitive situation of 275 participants over 65 years old, and the prevalence of MCI was 35.9 % (99/276) in a community in northwest China.The majority have completed their junior middle school education.The BMI was in the normal range, and the ratio of obesity to severe obesity was only 10.9 % (30/276).In terms of lifestyle and habits, most of them could keep a regular diet and do 40 min of exercise daily without smoking or consuming excessive alcohol.About half of the aging population complains of insomnia.In accordance with the previous study, sleep quality will decrease with aging (Brewster et al., 2018).Age, academic qualifications, finances, and exercising are all closely related to the development of MCI.Our research is in line with earlier studies in terms of age (Lu, 2017;Okello et al., 2009), educational attainment (Amieva et al., 2005), and daily exercise (van Praag et al., 2005;Baker et al., 2010;Suzuki et al., 2012), but there is a discrepancy in income that requires more research (Kim et al., 2016).Exosome synthesis involves the formation of specialized intracellular compartments, including MVB and ILV biogenesis (Zhang et al., 2019) where endosomal sorting complex required for transport (ESCRT) consists of four main complexes (ESCRT-0, -I, -II, and -III) works cooperatively to promote the whole process.Several ESCRT components (TSG101, Alix) have been identified in purified small extracellular vesicles (sEVs) from various cell types (Baietti et al., 2012).Also, some tetraspanins, such as CD63, CD9, have been regarded as key points in sEVs formation (Chairoungdua et al., 2010).For this study, we found that CD9 and TSG101 showed an increasing expression with age and MCI, reflecting aging or MCI may promote the secretion of exosome.As mentioned above, current studies have shown that exosomes may change due to aging related mechanisms (Lehmann et al., 2008;Takasugi et al., 2017;Takahashi et al., 2017), in consideration of exosome secretion, the specific condition of age-related disease should be considered.
Exosomes are the main source of miRNAs in human circulating blood (Zhang et al., 2015;Boon and Vickers, 2013).The profiling of miRNAs is different among diseases, which in turn could reflect the real state of the disease (Thery et al., 2002).Numerous studies have discovered MCIspecific miRNAs with some diagnostic efficacy (Shi et al., 2020), but further research is still needed to determine the utility of exosomal miRNAs in MCI and the viability of using them for diagnosis.
Since only a few studies have concentrated on the non-invasive specific markers of MCI in the community-dwelling elderly who are not hospitalized or do not have acute diseases, the inclusion criteria for participants in this study are the community-dwelling elderly.According to our findings, miR-483-5p, miR-502-5p, and miR-132-3p relative expression levels in plasma exosomes of the MCI group were higher than those of either the younger control group or the age-matched control group, or both groups, and the differences were statistically significant.The implications of our research extend beyond the laboratory, hinting T. Liu et al. at a potentially transformative approach for diagnosing MCI.The elevated and stable levels of miR-483-5p and miR-502-5p in plasma exosomes present an opportunity for an accessible, cost-effective, and minimally invasive diagnostic method.Unlike traditional clinical examinations, the use of these biomarkers offers a novel avenue for early MCI detection, potentially facilitating timely interventions and personalized treatment strategies.Incorporating plasma exosomal miRNAs into diagnostic protocols could significantly enhance our ability to identify individuals at risk of MCI, providing a valuable complement to existing clinical assessments and offering a practical solution for widespread and routine screening in aging populations.
This study encountered from several shortcomings.First, a relatively limited number of older persons were registered in the scale survey, who were mostly from metropolitan communities.To boost confidence, the follow-up study should raise the sample size even more.Additionally, the distinction between amnesic and non-amnesic MCI could not be made using verified specific exosomal miRNAs for the diagnosis of MCI.Therefore, additional research should concentrate on various MCI kinds according to the MCI's sophistication.For the confirmation of our findings and the mechanism of exosomal miRNA in brain aging, more research is required.
The findings of this study indicated that for senior people with lower levels of education, poorer incomes, and a lack of daily activity, effective intervention measures should be implemented as soon as possible.Elderly people should be encouraged to engage in daily, appropriate physical activity as well as simple, effective health education to improve their quality of life and delay cognitive deterioration.Second, miR-483-5p and miR-502-5p, which are derived from plasma exosomes and may be used as promising noninvasive biomarkers, are maintained at high levels in elderly people with MCI.
Future experimental investigations employing bioinformatics analyses are essential to validate the diagnostic significance of circulating miRNAs.Additionally, these studies should elucidate the regulatory mechanisms governing the involvement of these miRNAs in the pathogenesis of MCI in elderly patients.a-d.Fold changes of miR-483-5p, miR-502-5p, miR-132-3p and miR-206, in exosome.e. Receiver operating characteristic curves for miR-483-5p and miR-502-5p to discriminate MCI from age-matched control.

Fig. 1 .
Fig. 1.Network of enriched terms.Each color of the pie chart represents a combination of gene lists, where the size of a slice represents the percentage of genes under the term that originated from the corresponding gene list, allowing for easy identification and comparison of enriched terms across miRNA-132-3p, miR-206, miR-483-5p and miR-502-5p.Nodes in the network represented individual terms, and edges indicated the degree of term similarity, where terms with a similarity >0.3 are connected by edges.

Fig. 2 .
Fig. 2. Characterization of exosome plasma sample.a. Exosomal markers Alix, TSG101, CD9 and the markers in consistent supernatant were analyzed by Western blot.b.Morphology and size of exosome by TEM (Scale bar = 100 nm).

Fig. 3 .
Fig. 3. Comparison of exosome release among health control and MCI patients.a. Expression of Alix, TSG101, and CD9 by Western blot, identical level of plasma protein was verified by staining of Coomassie blue.b-d.Densitometry analysis of Alix, TSG101 and CD9.(n = 3 replications, one-way ANOVA with Newman Keuls multiple comparison test, bar graphs show Mean ± SEM. ns indicates no significant difference among groups).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1
miRNA qPCR primers used in this study.

Table 2
Demographic and clinical characteristics of enrollment.

Table 3
Demographic and clinical characteristics of participants.