The Eminent Role of microRNAs in the Pathogenesis of Alzheimer's Disease

Alzheimer's disease (AD) is an irrevocable neurodegenerative condition characterized by the presence of senile plaques comprising amassed β-amyloid peptides (Aβ) and neurofibrillary tangles mainly comprising extremely phosphorylated Tau proteins. Recent studies have emphasized the role of microRNAs (miRNAs) in the development of AD. A number of miRNAs, namely, miR-200a-3p, miR-195, miR-338-5p, miR-34a-5p, miR-125b-5p, miR-132, miR-384, miR-339-5p, miR-135b, miR-425-5p, and miR-339-5p, have been shown to participate in the development of AD through interacting with BACE1. Other miRNAs might affect the inflammatory responses in the course of AD. Aberrant expression of several miRNAs in the plasma samples of AD subjects has been shown to have the aptitude for differentiation of AD subjects from healthy subjects. Finally, a number of AD-modifying agents affect miRNA profile in cell cultures or animal models. We have performed a comprehensive search and summarized the obtained data about the function of miRNAs in AD in the current review article.


INTRODUCTION
Alzheimer's disease (AD) is an irrevocable neurodegenerative condition with a progressive course, and it is the chief reason for dementia in the elderly (Prince et al., 2013). AD is characterized by pervasive cognitive defects and memory deficits, leading to the dependence of the majority of AD patients on others for their routine activities. From a pathological point of view, AD is defined by the presence of senile plaques comprising amassed β-amyloid peptides (Aβ) and neurofibrillary tangles mainly comprising extremely phosphorylated Tau proteins (Ballard et al., 2011). The most accepted hypotheses for the development of AD are based on these two main pathological events [i.e., Aβ accumulation and Tau accumulation (Wang et al., 2019a)]. The amyloid precursor protein is converted to Aβ through consecutive enzymatic reactions catalyzed by β-secretase (BACE1) and γ-secretase (containing presenilin 1 and presenilin 2) (Querfurth and LaFerla, 2010). Recent studies have emphasized the role of microRNAs (miRNAs) in the development of AD (Wang et al., 2019a). These ∼22 nucleotide transcripts post-transcriptionally regulate the expression of several target genes through binding with 3' UTR and destructing the target transcript or reducing its translation (O'Brien et al., 2018). Sequence complementarity mainly regulates the miRNA/mRNA interactions leading to the ability of one miRNA to target several genes and the possible regulation of one gene by multiple miRNAs. Therefore, miRNAs are potential means for investigating multifactorial disorders such as AD (Iqbal and Grundke-Iqbal, 2010). A leading investigation in this regard has examined the number of brain-associated miRNAs expressed in the human hippocampus specimens obtained from fetal, adult, and AD patients, revealing misregulation of certain miRNAs in the AD brain and their possible contribution to the pathological processes of this disorder (Lukiw, 2007). Dysregulation of other miRNAs has also been verified in multiple studies, and the underlying mechanisms of their contribution in AD have been identified in some cases. We have performed a comprehensive search and summarized the obtained data about the function of miRNAs in AD in this review article.

DYSREGULATED MIRNAS IN AD
Dysregulation of miRNAs has been demonstrated in human AD subjects or animal models of AD. Moreover, several researchers have induced or suppressed the expression of some miRNAs in the cell/animal models of AD to appraise their function in the progression of AD. In a rat model of AD caused by the administration of Aβ 25−35 into the brain, downregulation of SOX6 and over-expression of miR-129-5p have shortened the dormant escape period and enhanced the time of crossing platforms, repairing the pathological damage, blocking neuronal apoptosis, and decreasing inflammation. Based on the protective effects of miR-129-5p against nerve damage and inflammation, miR-129-5p has been suggested as a candidate for therapeutic options against AD, as it acts to suppress SOX6 (Zeng et al., 2019). Expression of miR-200a-3p has been shown to be repressed in animal and cell models of AD. miR-200a-3p can suppress cell apoptosis, inactivate Bax/caspase-3 axis, and decrease Aβ 1−42 and tau phosphorylation in cell experiments. Mechanistically, these effects are mediated through the modulation of translocation of BACE1 and PRKACB. Taken together, the neuroprotective impact of miR-200a-3p is accomplished by inhibition of BACE1 expression and subsequent suppression of Aβ production as well as reduction of PKA expression and Tau phosphorylation (Wang et al., 2019b). miR-455-3p has been shown to bind with 3' UTR of APP gene to decrease its expression and reduce expression of mitochondrial fission proteins (Kumar et al., 2019). Mutant APP cells that show expression of miR-455-3p exhibit upregulation of synaptic genes. Over-expression of miR-455-3p in mutant APP cells reduces the number of mitochondria and increases the size of the mitochondria. Taken together, miR-455-3p controls APP processing and protects against mutant APPassociated mitochondrial dysfunction and synaptic anomalies in AD (Kumar et al., 2019). Expression of miR-455-3p has been shown to be increased in postmortem brain samples, fibroblasts, and plasma samples of patients with AD compared with controls Abbreviations: AD, Alzheimer's disease; microRNA, miRNA; Aβ, β-amyloid peptides; BACE1, β-secretase; EOAD, early-onset Alzheimer's disease; CNV, copy number variant; SNP, single nucleotide polymorphism; AChE, Acetylcholinesterase; iNOS, Inducible nitric oxide synthase; ROS, reactive oxygen species; MDA, Malondialdehyde; MAPK, mitogen-activated protein kinase; SOD, Superoxide dismutase; GSH-Px, glutathione peroxidase. (Kumar et al., 2017;Kumar and Reddy, 2018). As a primary event, expression of miR-409-5p has been decreased in an APP/PS1 double transgenic mice model of AD. Over-expression experiments have shown that this miRNA has a harmful impact on neurite outgrowth, reduces neuron survival, and quickens the progression of Aβ 1−42 -associated pathologic events (Guo et al., 2019). In line with the observed downregulation of miR-409-5p in APP/PS1 AD model, Aβ 1−42 peptide has been shown to downregulate miR-409-5p levels. A luciferase study has shown that Plek is a target of miR-409-5p (Guo et al., 2019). Ectopic expression of miR-409-5p has induced neurotoxic effects and interferes with neuron survival and differentiation, while Plek upregulation could partly protect the neurite outgrowth from these toxic effects. Taken together, reduction of miR-409-5p expression in the early stages of AD might be a self-protective response to lessen the synaptic injury induced by Aβ (Guo et al., 2019). miR-132 is another downregulated miRNA in AD. Experiments in a rat model of AD have shown upregulation of AChE, iNOS, ROS, MDA, MAPK1, and p-MAPK1 and downregulation of SOD, GSH-Px, and miR-132. Over-expression of miR-132 has reversed these markers demonstrating the role of this miRNA in the suppression of hippocampal iNOS expression and oxidative stress through reduction of MAPK1 levels (Deng et al., 2020). However, expression of this miRNA has been demonstrated to be reduced in neurally-originated plasma exosomes of AD subjects (Cha et al., 2019). Table 1 shows the summary of studies that reported decreased levels of miRNAs in AD.
Although several studies have reported downregulation of miR-132 in AD (Wong et al., 2013;El Fatimy et al., 2018;Cha et al., 2019;Deng et al., 2020), Liu et al. have reported high levels of miR-132 in patients with mild cognitive impairment and AD vs. normal individuals. They have shown the impact of miR-132 upregulation in the induction of apoptosis in neurons through increasing Bax/Bcl-2 ratio . Moreover, they have reported that miR-132 increases Tau phosphorylation and expression levels of Rb, Histone H1, and CDK-5. Collectively, they have suggested that miR-132 participates in AD by controlling cell apoptosis and the GTDC-1/CDK-5/Tau phosphorylation axis . In addition to GTDC-1, miR-132 is also known to regulate the expression of synaptic proteins via complement C1q (Xu et al., 2019). Similarly, expression of miR-132 has been shown to be decreased in AD-derived plasma exosomes (Cha et al., 2019). miR-128 has also been over-expressed in the brain samples of AD patients . Experiments in AD mice have demonstrated parallel upregulation of miR-128 and downregulation of PPARγ in the cerebral cortex. The interaction between these two transcripts has been validated through functional assays. miR-128 silencing has suppressed AD-like features, amyloid plaque creation, Aβ production, and inflammation in AD mice through upregulating PPARγ . miR-425-5p is another upregulated miRNA in patients with AD and the cellular model of AD. Upregulation of miR-425-5p has induced cell apoptosis, stimulated expression of GSK-3β, and enhanced tau phosphorylation through targeting HSPB8 (Yuan et al., 2020). miR-146a is also upregulated in AD and participates in the pathogenesis of this condition via targeting Lrp2 and inhibiting the Akt signaling pathway, modulating ROCK1 expression and decreasing Tau phosphorylation, and influencing inflammatory responses via modulation of IRAK-1 (Cui et al., 2010;Wang et al., 2016). Insulin and liver X receptor (LXR) activators have been shown to increase the miR-7-1 levels. Expression of this miRNA has changed within the brains of diet-induced obese animals as well as AD patients, which is in parallel with the downregulation of its target genes IRS-2 and IDE. Upregulation of miR-7 has enhanced extracellular Aβ levels in neurons and interfered with the eradication of Aβ by microglia. Collectively, insulin can act via the HNRNPK-miR-7 cascade to post-transcriptionally affect metabolic pathways in AD (Fernández-de Frutos et al., 2019). Table 2 lists upregulated miRNAs in AD. Figure 1 demonstrates the function of a number of miRNAs in the pathogenesis of AD.

PROGNOSTIC AND DIAGNOSTIC ROLE OF MIRNAS IN AD
The prognostic role of miRNAs in AD has been assessed in a single study. Xie et al. have evaluated serum levels of miR-206, miR-132, BDNF, and SIRT1 in a cohort of patients with amnestic mild cognitive impairment at baseline and after 5year follow-up. Their results have shown higher levels of miR-206 in patients who converted to AD both at the baseline and after 5-year follow-up. However, miR-132 levels have been statistically similar between the conversion and non-conversion groups at both time points. Based on the Kaplan-Meier analysis, AD conversion has been correlated with over-expression of miR-206. In addition, multivariate Cox regression analysis has shown the suitability of serum miR-206 and its target BDNF as indicators of AD conversion (Xie et al., 2017). The diagnostic role of several miRNAs has been appraised in AD. For instance, downregulation of miR-433 and miR-133b in serum samples could precisely differentiate between AD subjects and normal persons (Yang et al., 2019b;. Moreover, the expression profile of the former miRNA in the cerebrospinal fluid (CSF) has appropriate diagnostic power for distinguishing AD patients . The most astonishing results have been obtained for miR-34c. Expression of miR-34c has been increased in both cellular and plasma constituents of blood specimens of AD patients. The area under the receiver operating characteristic curve has been estimated to be 0.99. Moreover, expression of miR-34c has been inversely correlated with mental performance, as described by the mini-mental state examination. miR-34c has also been shown to affect the expression of numerous genes being involved in neuron survival and oxidative processes (Bhatnagar et al., 2014). Expression levels of miR-132 and miR-212 in neural-derived extracellular vesicles have been demonstrated to differentiate patients with AD from healthy subjects, yet their aptitude in identifying both AD and mild cognitive impairment as different from a healthy status has not been suitable (Cha et al., 2019). Table 3 summarizes the outlines of various studies that have reported on the diagnostic value of miRNAs in AD.   , and miR-339-5p (Long et al., 2014) are decreased in patients with Alzheimer's disease. These miRNAs bind with the 3' UTR of BACE1 and decrease its expression. Therefore, the downregulation of these miRNAs leads to the upregulation of BACE1. In addition, expression levels of some APP-binding miRNAs namely miR-101 (Vilardo et al., 2010), miR-153 (Liang et al., 2012), miR-16 (Liu et al., 2012), miR-384 (Liu et al., 2014b), miR-31 (Barros-Viegas et al., 2020), miR-193b (Liu et al., 2014a, and miR-455-3p (Kumar et al., 2019) is decreased in patients with Alzheimer's disease. (B) Tau phosphorylation leads to defects in microtubules and induction of neurofibrillary tangles which result in neuron death. miR-138 and miR-425-5p are increased in Alzheimer's disease. These miRNAs regulate the expression of GSK-3β and enhance Tau phosphorylation (Wang et al., 2015b;Yuan et al., 2020). In addition, downregulation of miR-132 and upregulation of miR-125b and miR-922 leads to Tau hyperphosphorylation Salta et al., 2016;Ma et al., 2017).

MIRNA POLYMORPHISMS AND RISK OF AD
Boscher et al. have screened a larger cohort of early-onset AD (EOAD) patients who did not have autosomal dominant mutations for the presence of genetic polymorphisms. They have recognized 86 copy number variants (CNVs) in miRNAcoding genes, 31 of them being only present in EOAD cases. Duplication of the MIR138-2 locus has been one of these CNVs. Based on the role of miR-138 in Aβ production and tau phosphorylation, this CNV might be implicated in the risk of EOAD (Boscher et al., 2019). Functionally, miR-138 upregulation enhances Aβ synthesis and tau phosphorylation through modulation of GSK-3β and FERMT2 (Boscher et al., 2019). Other studies have demonstrated the role of rs2910164 of pri-miR-146a, rs57095329 of miR-146a, and rs2291418 of miR-1229 precursor in conferring risk of AD (Table 4). Zhang et al. have scanned the coding region of pri-miR-146a in AD patients. Among the four single nucleotide polymorphisms (SNPs) located in this genomic region, rs2910164 has been identified as a risk locus for AD as the C allele of this SNP has enhanced risk of AD. Notably, this variant has been shown to reduce the expression of mature miR-146a-5p, releasing TLR2 from its inhibitory effects. Moreover, cell line studies have shown the impact of the C allele on upregulation of expression of TNF-α after induction with βamyloid. Therefore, this SNP might predispose patients to AD by disturbing the production of mature miRNA and influencing the activity and expression level of TLR2 (Zhang et al., 2015). Cui et al. have analyzed the genotype and allele frequencies of rs2910464 and rs57095329 of miR-146a and have reported that   Analysis of GWAS data on late-onset AD --rs2291418 was associated with AD risk. An allele of rs2291418 was correlated with an increased miR-1229-3p expression that targets an AD-related gene, SORL1, so can have an important role in AD. Ghanbari et al., 2016 the AA genotype of the former SNP increases susceptibility to AD and results in cognitive reduction in the affected individuals. Contrary to the previously mentioned study by Zhang et al., the risk genotype has been associated with higher levels of miR-146a in the PBMCs of control subjects and has exerted more robust effects on IL-6 and IL-1β synthesis following stimulation with LPS (Cui et al., 2014). Finally, in a genome-wide association study, Ghanbari et al. have detected an association between rs2291418 in the miR-1229 precursor and risk of AD. The risk allele of this SNP has been shown to increase the expression of miR-1229-3p, thus decreasing the expression of SORL1, an ADassociated gene. In addition, among more than 42,000 variants in miRNA-binding regions, 10 variants in the 3' UTR of nine genes have been associated with this disorder; among them has been rs6857, which enhances the miR-320e-mediated modulation of PVRL2 expression (Ghanbari et al., 2016).

EFFECTS OF HERBAL/CHEMICAL AGENTS ON THE EXPRESSION OF MIRNAS IN THE CONTEXT OF AD
Osthole, the active component of the fruits of the genus Cnidium moonnieri (L.) Cussion has been shown to affect the AD course via modulation of miRNAs expression. Lin et al. have shown miR-101a-3p as the main affected miRNA by osthole. APP has been identified as the target of miR-101a-3p. Osthole has enhanced the learning and memory aptitude in an animal model of AD, and it has inhibited APP levels by promoting the expression of miR-101a-3p (Lin et al., 2019). Other studies have verified the effects of Osthole on the expression of miR-9 (Li et al., 2016. Functionally, osthole enhances the viability of neurons, decreases apoptosis of these cells, and reverses the decline of synaptic proteins in APP-expressing cells by affecting miR-9 expression and consequently decreasing CAMKK2 and p-AMPKα levels (Li et al., 2016). Additionally, osthole has pro-survival effects in APP-expressing neural stem cells through suppression of the Notch pathway . Moreover, Berberine has been shown to enhance proliferation and attenuate neuron apoptosis via regulation of miR-188/NOS1 molecular cascade (Chen et al., 2020b). Treatment of Aβtreated murine microglia and neuroblastoma cells with this substance or upregulation of miR-188 in these cells has accelerated cell proliferation and suppressed caspase-3 activity and apoptosis (Chen et al., 2020b). Finally, exmedetomidine has been demonstrated to accomplish neuroprotective effects and enhance cognitive function in an animal model of AD by modulating the miR-129/YAP1/JAG1 cascade . Table 5 shows the effects of different AD-modifying compounds on the expression of miRNAs.

DISCUSSION
Numerous studies have demonstrated abnormal expression of miRNAs in AD subjects or cell/animal models of AD. However, each miRNA has been the subject of expression and functional assays in few independent studies. miR-146 has been among the miRNAs most assessed in the context of AD, as its expression levels, functions, and polymorphisms have been assessed in association with AD. miR-9 is another AD-associated miRNA whose expression has been altered following treatment of APPexpressing cells with anti-AD substances. In some cases, altered expression of a certain miRNA is regarded as a part of a self-protective process. For instance, the reduction of miR-409-5p expression in the early stages of AD might be associated with lower Aβ-induced synaptic injury (Guo et al., 2019). Similarly, upregulation of miR-200b and miR-200c has protective effects against AD-associated neurotoxicity (Higaki et al., 2018). However, in most cases, an aberrant miRNA signature directly participates in the pathogenesis of AD. miRNAs partake in the pathobiology of AD through various mechanisms, including the regulation of BACE1 activity. miR-200a-3p, miR-195, miR-338-5p, miR-34a-5p, miR-125b-5p, miR-132, miR-384, miR-339-5p, miR-135b, miR-425-5p, and miR-339-5p are among the miRNAs whose interaction with BACE1 has been verified in different investigation. Other miRNAs, such as miR-129-5p, miR-22, and miR-206, might affect the inflammatory responses in the course of AD. Moreover, a number of miRNAs, such as miR-326, miR-338-5p, miR-124-3p, miR-34a, miR-326, and miR-98, modulate apoptotic pathways in neurons, thereby affecting the AD course via this route. Tau phosphorylation can be modulated by some miRNAs, namely, miR-200a-3p, miR-326, miR-124-3p, miR-146a, miR-425-5p, and miR-132. Expression of miR-132 has been assessed by several investigations with most of them reporting its downregulation in AD (Wong et al., 2013;El Fatimy et al., 2018;Cha et al., 2019;Deng et al., 2020). Yet, Liu et al. have reported over-expression of miR-132 in patients with mild cognitive impairment and AD vs. normal individuals . Abnormal levels of miRNAs in serum or CSF samples have been shown to distinguish AD subjects from normal subjects, indicating their suitability as disease biomarkers. However, these studies have not been validated in independent cohorts. miR-103, miR-126, miR-93, miR-29, miR-424, and miR-181 are among AD-associated miRNAs with biomarker potential whose application as disease biomarkers has been validated in other disorders (So et al., 2020).
Animal studies have shown promising results regarding the impact of miRNA modifications on the course of AD. However, based on the unavailability of brain tissue for therapeutic interventions in human subjects, identification of appropriate transport mechanisms for delivery of anta-/ago-miRs to this tissue is an important issue.
The anti-AD effects of Osthole, Tiaoxin Recipe, Berberine, and Dexmedetomidine have been shown to be exerted through modulation of a number miRNAs, such as miR-101a-3p, miR-9, miR-34a, miR-188, and miR-129, emphasizing further the impact of miRNAs in the progression of AD. However, these results should be verified in human subjects as well.
Few studies have shown the association between miRNA CNVs/ SNPs and the risk of AD. However, these results have not been verified in different ethnic groups. Re-assessment of the results of genome-wide association studies with a focus on noncoding regions might lead to the identification of further risk loci for this multifactorial condition.
Finally, a limitation of several functional investigations in this field is that they have assessed miRNA functions in cell lines such as HEK293 and SH-SY5Y, which are not true models of AD.

AUTHOR CONTRIBUTIONS
MT and SG-F wrote the draft and revised it. MS, MH, and MG collected the data, designed the tables, and figures. All authors contributed to the article approved the submitted version.