MicroRNA-455-3P as a peripheral biomarker and Therapeutic Target for Mild Cognitive Impairment and Alzheimer’s disease

Abstract

MicroRNAs are small non-coding RNAs evolutionary conserved molecules. They regulate cellular processes, including RNA silencing, post-translational gene expression and neurodegeneration. MicroRNAs are involved with human diseases such as cancer, Alzheimer’s disease (AD) and others. Interestingly, cerebrospinal fluids (CSF) and the blood of AD patients have altered expressions of many RNAs, which may serve as potential peripheral biomarkers. The intensive investigation from our lab revealed that MicroRNA-455-3p (miR-455-3p) is a strong candidate as a potential biomarker and therapeutic target for AD. Several genes implicated in the pathogenesis of AD are directly targeted by miR-455-3p. Several years of our lab research revealed that miR-455-3p regulates important physiological processes associated with AD, such as the processing of the amyloid precursor protein (APP), TGF-β signaling, the regulation of oxidative stress, mitochondrial biogenesis, and synaptic damages. The expression of miR-455-3p in mild cognitive impaired subjects and AD patients pointed out its involvement in AD progression. Recently, our lab generated both transgenic and knockout mice for miR-455-3p. Interestingly miR-455-3p transgenic mice showed superior cognitive learning, improved memory and extended lifespan compared to age matched wild-type mice, whereas mir-455-3-p knockout mice showed cognitive decline and reduced lifespan. Information derived from mouse models further demonstrated the advantageous impact of miR-455-3p on dendritic growth, synaptogenesis, and mitochondrial biogenesis in preventing the onset and progression of AD. The identification of miR-455-3p as a biomarker was suggested by its presence in postmortem AD brains, B-lymphocytes, and fibroblasts. Our hypothesis that miR-455-3p could be a peripheral biomarker and therapeutic target for AD.

Introduction

The expression of genetic information involves transcription of ribonucleic acid (RNA) molecules from deoxyribonucleic acid (DNA) and their role in protein biosynthesis. Mature messenger RNAs are linear entities with 5′ and 3′ termini that reflect start and stop of the RNA polymerase on the DNA template (Jacob and Monod, 1961). Transcription process of the whole eukaryotic genome also produces a plethora of non-protein-coding RNA species with complicated overlapping patterns of expression and control. Later numerous regulatory RNAs of all shapes and sizes have been discovered (Amaral et al., 2008; Ponting et al., 2009). Noncoding transcripts are classified as either housekeeping or regulatory. Housekeeping noncoding RNAs (ncRNA), including as ribosomal, transfer, small nuclear, and small nucleolar RNAs. Short regulatory noncoding RNAs include microRNAs (miRNA), small interfering RNAs, and Piwi-associated RNAs (Carthew and Sontheimer, 2009; Malone and Hannon, 2009). In most instances, ncRNAs are directly involved in the regulation of most cell processes, including epigenetic control, gene transcription, translation, RNA turnover, chromosomal organization, and genome defense, with key roles in cellular developmental and proliferation programs (Cech and Steitz, 2014; Huntzinger and Izaurralde, 2011; Morris and Mattick, 2014; Rinn and Chang, 2012; Sabin et al., 2013).

miRNA molecules have emerged as key regulators in the expression and function of eukaryotic genomes over the last 2 decades. This regulation, mostly inhibitory in nature, can occur at the most fundamental levels of genome function, such as chromatin structure, chromosome segregation, transcription, RNA processing, RNA stability, and translation. miRNAs inhibit the expression of mRNA targets by promoting translational repression, and mRNA degradation. Translational repression is thought to occur in four different ways: inhibition of translation initiation, the suppression of translation elongation, co-translational protein degradation, and premature termination of translation (Eulalio et al., 2008; Wu and Belasco, 2008). In animals, target degradation begins with rapid deadenylation, which is typically followed by decapping and subsequent degradation of the mRNA, but in plants, target degradation begins with endonucleolytic cleavage catalyzed by the argonaute proteins (Krol et al., 2010; Voinnet, 2009). A mature miRNA typically attaches to the 3′ untranslated region (UTR) of the designated messenger RNA (mRNA) as a part of the RNA-induced silencing complex (RISC) and inhibit the translation process (Figure. 1) (Iwakawa and Tomari, 2015). The fundamental determinant of target recognition is a brief sequence complementarity between the miRNA seed sequence (nucleotides 2–7) and the targeted mRNA (Lewis et al., 2005).

Figure 1:

Overview of miRNA biogenesis. miRNAs are transcribed by RNA polymerases II or III as pri-mRNA. primary miRNAs (pri-miRNAs) are recognized and excised by the microprocessor complex, composed by DiGeorge Syndrome Critical Region 8 (DGCR8) and Drosha, into a hairpin-shaped precursor-miRNAs (pre-miRNAs) via the canonical pathway in the nucleus. The pre-miRNA is exported to the cytoplasm in an Exportin 5/RanGTP-dependent manner. In the cytoplasm, dicer cleaves pre-miRNAs into tiny double-stranded miRNAs. One of the strands of the miRNA duplex is incorporated into the Argonaute (AGO) family of proteins to form a miRNA-induced silencing complex (miRISC). The miRISC complex then mediates the recognition of the target mRNA at the 3′ untranslated region, which results in mRNA instability or translational repression. The gene regulatory power of cytoplasmic miRISC typically culminates via induction of translation inhibition and mRNA poly(A) deadenylation.

Depending on their origin and expression patterns, certain miRNAs are highly tissue specific and restricted to specific cellular niches, whereas other miRNA families are expressed throughout the human body (Ambros, 2004; Bartel, 2004; Ludwig et al., 2016). In several occasions, miRNAs are encapsulated in a variety of structures, including exosomes, microvesicles, and apoptotic bodies, which release them into the extracellular space to regulate intracellular communication (Kosaka et al., 2013; Turchinovich et al., 2013). Recently scientists are considering circulating miRNA as potential biomarkers for cancer, ageing and neurological disorders (Kosaka et al., 2010; Kumar et al., 2017a; Kumar et al., 2017b; Olivieri et al., 2017).

Alzheimer’s disease (AD) is a neurological disorder that progresses over time and is heterogeneous. The hallmark of AD is memory loss, cognitive function impairment, and changes in behavioral and personality traits (LaFerla et al., 2007; Mattson, 2004; Reddy et al., 2010, Oliver and Reddy, 2019; Morton et al., 2021). Early-onset familial AD caused mostly by genetic mutations while late-onset sporadic AD is induced by ageing and other lifestyle factors (Amakiri et al., 2019; Sehar et al., 2022; Rawat et al., 2022). Mutations in amyloid precursor protein (APP), presenilin 1 (PS1), presenilin 2 (PS2), apolipoprotein E4 (APOE4), srotilin related receptor 1, clusterin, complement component receptor 1, CD2 Associated Protein (CD2AP), Aph-1 Homolog A (APHA1), and membrane-spanning 4-domains subfamily A (MS4A4/MS4A6E) genes have been reported in different AD patients (Mao and Reddy, 2011).

AD is marked by accumulation of intracellular neurofibrillary tangles (NFTs) and extracellular amyloid-β (Aβ) plaques in brain leading to synaptic damage, proliferation of reactive astrocytes and activated microglia, an age-dependent imbalance in hormones, and structural and functional changes in mitochondria (DeKosky et al., 1996; Du et al., 2010; Reddy, 2006; Reddy and Beal, 2008; Reddy et al., 2012; Swerdlow, 2011; Tampellini and Gouras, 2010; Terry et al., 1991). Although a great deal of work has been done in understanding the pathophysiology of AD, there are currently no identifiable biomarkers, medications, or other interventions that can stop AD or decrease its progression. The hunt for novel, non-invasive, peripherally available, early detectable biomarkers associated with AD and diseases related to Alzheimer’s disease (ADRD) is still progressing. Though many miRNAs that regulate AD genes to regulate AD pathogenesis and are involved in AD-related pathways have been found (Chopra et al., 2021; Kim et al., 2015; Kumar and Reddy, 2016; Long and Lahiri, 2011; Long et al., 2019; Ma et al., 2017; Vilardo et al., 2010), none of them can be definitively identified as an established biomarker for AD detection.

We highlight the relevance of miR-455-3p in AD in this review, mostly based on our lab’s (Reddy lab) findings, which are corroborated by other researchers. These findings lead us to establish miR-455-3p as a peripheral biomarker that can be found in the blood, CSF, fibroblast cells, and postmortem brains of AD patients.

1.1. Expression and biogenesis of miRNA-455-3P

MiRNA-455, with a highly conserved expression pattern, is present as non-coding RNA in most of the species from Chordata to Mammalia (Kumar and Reddy, 2018a). MiRNA-455 precursor sequence is transcribed by the intron 10 of human COL27A1 gene (collagen type XXVII alpha 1 chain). This precursor has 96 base pairs and is originally located at human chromosome 9 at locus 9q32 (Kumar and Reddy, 2018a). Human miR-455-3p has two different isoforms: miR-455-3p and miR-455-5p. miR-455 has key roles in different types of cancer ; human non-small cell lung cancer (Gao et al., 2018), oral squamous cancer cells (Cheng et al., 2016), renal cancer (Yamada et al., 2018), skin cancer (Shoshan et al., 2015), colorectal cancer (Zheng et al., 2016), hepatocellular carcinoma (Qin et al., 2016), breast cancer (Li et al., 2017b), pancreatic cancer (Zhan et al., 2018), and prostate cancer (Arai et al., 2019). In this review, we focus on its role in AD.

For more than eight years, the Reddy laboratory has been investigating miRNAs in ageing and age-related neurodegenerative disease such as AD. Earlier we reviewed the potential of circulating miRNAs as a peripheral biomarker for AD and their biological relevance in AD diagnosis, progression, and therapeutics. We found several probable miRNA candidates in AD (Kumar and Reddy, 2016). miR-455-3p got our attention when we found high expression in a global microarray analysis of serum samples collected from AD patients (Kumar et al., 2017b). Another research group also reported upregulation of miR-455-3p in hippocampus of AD patients compared to healthy controls (Lau et al., 2013). Further, our lab verified and validated the level of miR-455-3p in different kinds of AD samples (Kumar and Reddy, 2018b). We reported an elevation of MIRNA 455-3-p level on AD postmortem brains, AD fibroblasts, AD B-lymphocytes, AD cell lines, and APP transgenic mice [40, 41] compared to the samples from healthy controls. Moreover, we investigated the prospective role of miR-455-3p in AD biogenesis in in-silico, in vitro and in vivo models.

Here, we shed light to the significance of our findings and connection of miR-455-3p in the pathology, progression and modulation of MCI and AD. First, we discuss possible molecular and cellular events where miR-455-3p is offering a protective role in AD pathogenesis. In the later sections, we discuss the superior features of miR-455-3p in mice models in terms of ageing and AD development. Considering all these findings, we emphasize that miR-455-3p as a solid peripheral biomarker to diagnose and identify AD at an early state.

1.2. Role of miRNA 455-3P in AD related molecular and cellular events

Key cellular features of AD include not only accumulation of NFTs and Aβ plaques but also proliferation of reactive astrocytes, loss of cholinergic neurons due to synaptic damage, loss of synaptic proteins, defective autophagy/mitophagy and structural and functional alteration of mitochondria. According to our lab findings and other scientific reports miR-455-3p is directly regulating to some of the above features while indirectly affecting the others (Kumar et al., 2021).

a. Amyloid precursor protein processing (APP) and Tau as targets for miRNA 455-3P

Our lab investigated the protective role of miR-455-3p against APP processing and Aβ formation (Kumar et al., 2019a). Initially, predictive consequential pairing of has-miR455-3p.1 and has-miR-455-3p.2 with complementary target genes was studied and the conserved miR-455-3p.2 binding site was found in 323 projected transcripts/human genes. Surprisingly, a good number of target genes are involved in AD pathogenesis such as APP, NGF, USP25, PDRG1, SMAD4, UBQLN1, SMAD2, TP73, VAMP2, HSPBAP1, and NRXN1 (Kumar and Reddy, 2018b). In-silico analysis was performed to understand the link of miR-455-3p with gene regulating AD pathogenesis (Kumar et al., 2019a). We analyzed miR-455-3p target genes utilizing various online miRNA bio-informatics algorithms; for example, diana-microt, microrna.org, mirdb, rna22-has, targetminer, and targetscan-vert. It showed two binding sites of miR-455-3p at 3′ UTR of the APP gene (ENST00000346798.3) at sequence positions 522–528 and 3139–3145. The miR-455-3p binding sequence at the 3’UTR of APP mRNA was similar in the human and mouse genome. It suggests that miR-455-3p has a critical role in APP gene regulation affecting the expression level of APP genes. To validate these results, a luciferase reporter assay was conducted on a vector having a wild-type APP 3’UTR sequence and on a control vector. Luciferase activity was significantly decreased in the cells transfected with miR-455-3p mimics compared to control. These results define that the 3’UTR site of mRNA from the APP protein contains conserved binding sites for miR-455-3p (Figure 2). Moreover, in our qRT-PCR analysis, mRNA levels of APP gene were significantly reduced in the cells transfected with miR-455-3p mimics while strikingly upregulated in the cells transfected with the miR-455-3p inhibitors (Kumar et al., 2019a).

Figure 2:

miR455-3p has a critical role in APP gene regulation as miR-455-3p binds to two sequence locations in the 3′ UTR of the APP gene. It ultimately decreases the level of amyloid generation and accumulation in brain.

The protective role of miR-455-3p in AD was reveled via modulation of amyloid-β protein precursor (APP) and amyloid-β (Aβ) levels in mouse neuroblastoma cells. Overexpression of miR-455-3p construct reduces the expression of mutant APP cDNA and c-terminal fragments of APP (C99 and C83). Furthermore, miR-455-3p significantly reduced levels of Aβ (40) and (42) in mutant APP cells compared to control (Figure 2) (Kumar et al., 2019a) which ultimately explains the mechanistic role of miR-455-3p in AD progression.

Supporting our data, another article identified APP, BACE1 (Beta-site APP cleaving enzyme-1) and TAU as direct targets for miR-455-3p cause overexpression of miR-455-3p leading to a reduction in the expression of these genes in neuroblastoma cells (Swingler et al., 2022). In addition, they found an increase in the protein level of APP, BACE1 and TAU in the hippocampus of miR-455 null mice. Such findings reinforce the direct role of miR-455 in AD progression (Figure 2).

b. miR-455-3p and TGF-β signaling

In our lab miR-455-3p target genes were analyzed and surprisingly one TGFβ receptor, TGFBR3L, and two downstream regulators of TGFβ signaling pathway were identified namely SMAD2 and SMAD4 (Kumar and Reddy, 2018b). Supporting evidences were reported in other articles where they identified SMAD3 as a target gene of miR-455-3p (Liu et al., 2021) and showed that miR-455 downregulated TGFBR3 (Gharanei et al., 2020). In the TGF-β pathway, once bioavailable TGF-β comes into contact of the target cell, it binds with TGF-β type II receptors (TβRII) followed by a stable complex formation with TGF-β type I receptor (TβRI) (Massagué, 1998; Shi and Massagué, 2003). The activated ligand-receptor complex is then internalized via endocytosis (Chen, 2009). SMAD2 and SMAD3 are receptor-regulated effector proteins (R-Smads), which are phosphorylated by the activated TβRI, resulting in making a complex with SMAD4 (also known as co-SMAD) which ultimately activates the nuclear accumulation of SMAD complex (Massagué et al., 2005). Hence, by regulating the expression of SMAD2 and SMAD4 expression, miR 255-3-p might play a definitive role in TGF-β pathway in different aspects of cellular function (Figure 3).

Figure 3:

miR455-3p activates TGF beta signaling by regulating PAK2 expression,which ultimately ameliorates Alzheimer’s disease progression.

Several other reports have supported our claim that miR-455-3p has a critical role in TGF-β pathway. First, Swingler et al., found that miR-455-3p directly targets SMAD2, ACVR2B (Activin Receptor Type IIB) and CHRDL1 (Chordin-like 1) which could potentially modulate its functional impact on TGFβ signaling (Swingler et al., 2012). CHRDL1 gene encodes an antagonist of bone morphogenetic protein 4 (BMP4). As activin and BMP4 are essential component of TGFβ pathway, it indicates that miR-455-3p directly regulates this pathway in different probable routes in different cell functions. They also reported that the expression of miR-455-3p was regulated by TGFβ ligands during chondrogenesis. The upregulation of miR-455-3p were also reported in primary parenchymal lung fibroblasts after TGF-β1 stimulation (Ong et al., 2017). They also found NGF (nerve growth factor) and DLD (dihydrolipoamide dehydrogenase) genes as a target for miR-455-3p and these genes are involved in the TGF-β1-related biological processes and signaling pathway. On the contrary, downregulation of miR-455-3p upon TGF-β1 stimulation were reported in idiopathic pulmonary fibrosis (IPF) by another scientist group (Milosevic et al., 2012). This discrepancy could result from variations in the use of one donor versus several, culture techniques, and duration of TGF-β1 stimulation. Later, Zeng and colleagues found that TGF-β–induced protein ZEB1 (Zinc finger E-box-binding homeobox 1) is one of the target genes for miR-455-3p and through which it inhibited TGF-β signaling in a feedback loop mechanism (Zeng et al., 2019). Similar regulatory role of miR-455-3p via inhibiting ZEB1 expression was also reported during TGF-β1 induced signaling in endothelial to mesenchymal transition (Zhang et al., 2020). Another article suggested that miR-455-3p promoted the TGF/SMAD signaling pathway in chondrocytes. miR-455-3p inhibited the expression of P21-activated kinase 2 (PAK2) by directly targeting the 3′UTRs of PAK2 mRNA which ultimately promoted the R-SMAD activation (Figure 2) (Hu et al., 2019).

TGF-β1 signaling critically contributes to the proper functioning and protection of the brain in many ways. TGF-β1 shields neurons against harm caused by trophic factor deficiency, excitotoxins, hypoxia/ischemia, and Aβ aggregates (Caraci et al., 2008; Dhandapani et al., 2003). TGF-β1 also increases the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-xl and maintains mitochondrial membrane potential (Prehn et al., 1996). On the contrary, reduced expression of TGFβRII receptor and impairments of TGF-β-activated SMAD signaling in AD brain were reported in several occasions (Chalmers and Love, 2007; Lee et al., 2006; Tesseur et al., 2006; Ueberham et al., 2006) suggesting dysfunction of TGF-β pathway is probably a causal factor in AD progression. Hallmarks of AD pathogenesis like NFT accumulation (Chalmers and Love, 2007; Lee et al., 2006) and Aβ deposition (Wang et al., 2010) disrupt the TGF/SMAD signaling pathway in AD brain. Taken together, further investigation is necessary to completely understand the fascinating part that TGF/SMAD signaling controlled by miR-455-3p plays in the development or prevention of AD.

c. Role of miR455-3p in alleviating oxidative stress and cell survival in apoptosis

Our preliminary miR-455-3p mimic transfection studies provide crucial hints regarding their defensive functions in cell proliferation and viability. In our lab, we found that neuroblastoma cells treated with miR-455-3p mimics showed extended cell proliferation compared to the controls whereas cells transfected with miR-455-3p inhibitors did not show regular proliferation (Kumar et al., 2019a). In addition, we conducted apoptosis analysis utilizing Annexin V-FITC reagent and found treatment with miR-455-3p significantly reduced apoptotic cell death and increased level of cell survival. Zhang and colleagues also found that miR-455-3p increased cell proliferation, promotes cell survival in MC3T3-E1 osteoblast cells (Zhang et al., 2018). miR-455-3p also promoted cell proliferation in pancreatic cancer cells (Zhan et al., 2018) and in breast cancer cell lines (Li et al., 2017a). Therefore, miR-455-3p may target different genes regulating cell proliferation and cell survival.

Several genes (e.g., BAX, BMF, and HIF1) mediating oxidative stress were marked as target genes for miR-455-3p in our lab data (Kumar et al., 2019a). Individuals with MCI, AD and AD related dementia exhibit a notable degree of oxidative damage to their brains, which is linked to the aberrant build-up of Aβ and the formation of NFTs (Christen, 2000; Huang et al., 2016). A number of articles proposed that miR-455-3p protects cells from oxidative stress. miR-455-3p shields osteoblasts from oxidative stress by targeting HDAC2 (histone deacetylases 2) to trigger nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (AREs) signaling (Zhang et al., 2018). Moreover, Wang et al. reported that miR-455-3p played a protective role against high glucose-induced oxidative stress and cell injury by targeting Hmgb1 (high-mobility group box 1) gene (Wang et al., 2022). miR-455-3p also reduced apoptosis in chondrocyte through targeting PTEN (phosphatase and tensin homolog) gene (Wen et al., 2020) in PI3K/AKT pathway (Wen et al., 2020). In an oxygen and glucose deprivation (OGD) cell culture model, miR-455-3p alleviated oxidative stress related inflammatory responses by targeting SOX2-OT (sex-determining region Y-box 2 overlapping transcript) and TRAF6 (tumor necrosis factor receptor associated factor 6) genes (Gu et al., 2020). Strikingly, miR-455-3p protected neurons from oxidative injury by targeting small nucleolar RNA host gene 15 (SNHG15) and tumor protein p53 inducible nuclear protein 1 (TP53INP1) mRNAs (Fan et al., 2021). Taken together, we would like to suggest that miR-455-3p might have a distinctive role in mediating oxidative stress and inflammation in AD pathogenesis.

d. miR455-3p and mitochondrial biogenesis

Given the development of the AD are predominantly caused by structural and functional abnormalities in the mitochondria (Calkins and Reddy, 2011; Reddy, 2011), our lab evaluated the impact of miR-455-3p on the mRNA and protein levels of mitochondrial biogenesis genes (PGC1α, NRF1, TFAM). We found those were significantly increased in cells co-transfected with miR-455-3p and mutant APP relative to cells transfected with mutant APP alone. Further, we investigated the effect of miR-455-3p on mitochondrial dynamics genes. mRNA and protein levels of mitochondrial fission proteins (DRP1 and FIS1) were upregulated in the cells co-transfected with miR-455-3p and mutant APP cDNA. On the contrary, and fusion proteins (OPA1, Mfn1, and Mfn2) were decreased in the same experiment after miR-455-3p treatment. In addition, miR-455-3p also showed protective effects against mutant APP-induced mitochondrial fragmentations and repaired the abnormal mitochondrial dynamics, as captured in our electron microscopic analysis of neuroblastoma cells co-transfected with mutant APP cDNA and miR-455-3p (Kumar et al., 2019a). Supporting this, mitochondrial biogenesis and dynamics were improved in our miR-455-3p TG (transgenic) mice, while these were reduced in KO (knock out) mice what will be elaborated in later sections of this article. These findings lead us to the conclusion that miR-455-3p overexpression preserves normal mitochondrial activity by lessening the harmful effects of Aβ on mitochondria.

It is debated, nevertheless, how miR-455-3p controls mitochondrial activities. miR-455-3p targets hypoxia-inducible factor 1 α inhibitor (HIF1an) gene which has a key regulatory functions in mitochondrial biogenesis via interacting HIF1an- AMP-activated kinase α1 subunit (AMPK α1)-PGC1α signaling network (O’Hagan et al., 2009; Yun et al., 2019; Zhang et al., 2015). Through the inhibition of HIF1an-mediated AMPKα1 hydroxylation, miR-455-3p, activated AMPKα1 and triggered PGC1α phosphorylation leading to stimulation of mitochondrial biogenesis (Figure 4). Another hypothesis behind the protective role of miR-455-3p in mitochondria by targeting mitogen-activated protein kinase kinase 1 (MEKK1) gene (Wang and Shen, 2022), which is well known for the transition of mitochondrial permeability leading to apoptosis (Gibson et al., 2002; Mendoza et al., 2005). A binding correlation between miR-455-3p and MEKK1 was initially found in bioinformatics analysis and then validated by dual luciferase and RIP (RNA-Protein pull-down) assays. miR-455-3p specifically targeted and lowered MEKK1 expression also in in-vitro cell culture (Wang and Shen, 2022).

Figure 4:

Role of miR455-3p in mitochondrial biogenesis and synaptic architecture. miRNA 455-3p repress HIF1an expression and improves mitochondrial biogenesis. Our hypothesis is miRNA improved dendritic arbor and synaptic strength by positively regulating mitochondrial function and oxidative stress.

e. miR-455-3p enhanced dendritic and synaptic architecture

miR-455-3p has a significant role in promoting neuronal growth, supporting dendritic architecture, developing spines and augmenting synaptogenesis. At first, we observed a significant increase in synaptic proteins like synaptophysin and PSD95 in neuroblastoma cells (Kumar et al., 2019a) supporting its role in the regulation of synaptic activities. Moreover, miR-455-3p showed protective effects against AD related synaptic damages as synaptic protein levels were increased significantly in miR-455-3p co-transfected cells compared to the cells transfected with mutant APP alone. In our miR-455-3p TG mouse model, miR-455-3p overexpression increased neuronal populations, dendritic lengths and density, number of spines in hippocampal segments of TG mice brain (Kumar et al., 2021a). Furthermore, average synapse numbers were significantly upregulated in TG mice compared to WT. Findings from our TG and KO mice models are elaborated in the letter section of this article.

Several other articles reported the role of miR-455-3p in protecting neuronal survival against neurotoxicity. First, Yao et al., reported that miR-455 had a vital role in protecting neuronal cell death by downregulating TNF Receptor Associated Factor 3 (TRAF3) in cerebral ischemic stroke (Yao et al., 2016). Second, high levels of miR-455-3p reduces the propofol-induced neurotoxicity by suppressing the EphA4 (ephrin Type-A Receptor 4) expression in hippocampal neurons (Zhu et al., 2020). Third, miR-455-3p ameliorated hippocampal neuronal injury by conversely regulating programmed cell death 7 (PDCD7) expression (Gan and Ouyang, 2022). Though these findings strongly suggest its protective role in progression and pathogenesis of AD, further research is still required to understand the underlying mechanism(s).

f. miR-455-3p mice models showed superior cognitive ability and extended longevity

Elevated amount of miR-455-3p have been shown to increase synaptic activity, mitochondrial function and biogenesis, and decrease Aβ pathology, based on both in vitro and in vivo studies in our lab (Kumar et al., 2021b). The widely accepted models for examining the physiological processes and functions of miRNAs in the progression of AD are mice. Consequently, we present a thorough summary of the implications of miR-455-3p on cognitive behaviour, mitochondrial activity, and synaptic activity by investigating our miR-455-3p KO and TG mice models.

A number of AD mice models, including KO, knock-in, and TG models, have been employed to both produce therapeutic medications and comprehend the disease process from the point of birth to the end. These models allow researchers to look into the effects of gene products and their genetic mutations, and to evaluate anti-AD medications by crossing them with other TG and KO mice (Elder et al., 2010; Hall and Roberson, 2012). A innovative method for examining the advantageous or detrimental impacts of miRNAs in human diseases is the use of miRNA mouse models (Delay and Hébert, 2011; Pal and Kasinski, 2017). A single miRNA, such as miR-455-3p, can control several target proteins, which is why it is linked to a number of human diseases, such as chondrogenesis and cancer (Kumar and Reddy, 2019). Because of miR-455-3p’s important functions in AD and possible anti-AD benefits, the development of miR-455-3p mice models has become imperative. In addition to AD, miR-455-3p mice models could improve knowledge about and therapy for additional human disorders linked to miR-455-3p.

It has been discovered that miRNA molecules profoundly affected endogenous human genes and proteins on an epigenetic and genetic level (Bartel, 2004). Numerous miRNAs have been investigated in relation to AD, and their potential impacts on AD-related genes and proteins have been shown. In-depth investigation was hindered in the majority of miRNA studies due to the absence of appropriate mice models (Kim et al., 2015; Kumar et al., 2021b; Vilardo et al., 2010; Zhao et al., 2019). Numerous investigations used direct oral/nasal delivery of miRNA complexes or injections of miRNAs (Kumar et al., 2021b; Merhautova et al., 2016). The efficacy of these methods was restricted by various parameters, including the exogenous miRNA complex’s half-life within the body, immunological responses to miRNA-encapsulated particles, the stability of the miRNA complexes, and the route and dosage of miRNA administration. The creation of miRNA mice models, which offer comprehensive insights into the physiological mechanisms and therapeutic potential of particular miRNAs, is perfect for overcoming these obstacles and technological constraints (Kumar et al., 2019b; Kumar and Reddy, 2018b, 2019, 2021; Kumar et al., 2017b). Based on earlier studies into miR-455-3p and its prospective anti-AD capabilities, miR-455-3p KO and TG models were generated effectively by employing CRISPR/Cas9 knockout procedures and pronuclear injection of the miR-455-3p transgene into mice embryos (Figure 5). miR-455-3p expression levels were examined by immunoblotting, qRT-PCR, sequencing, and genotyping in TG and KO mice. In these animal models, the insertion or deletion of the miR-455-3p transgene did not result in notable phenotypic abnormalities (Kumar et al., 2021b).

Figure 5:

Unveiling the role of different mice models of miR-455-3p in diverse physiological processes.

Our study tracks non-KO and non-transgenic WT, TG, and KO mice from birth to the end of their lives, which provides an excellent explanation for the extinction of life span. According to survival data, age-matched transgenic mice outlived their non-transgenic WT siblings by 5 months, but KO mice outlived their WT siblings by 4 months (Kumar et al., 2021b). This explains why miR-455-3p overexpression increases longevity; put another way, miR-455-3p has brain-defending attributes. Given that naturally generated whole APP protein and mRNA levels were higher in KO mice and lower in miR-455-3p TG animals, the findings imply that reducing full-length APP may have a protective effect on the brain (Kumar et al., 2021b; Swingler et al., 2022). The significant rise in endogenous APP levels in KO mice’s brains suggests the value of using KO animals to investigate APP-related toxicity in AD. Crossing miR-455-3p TG/KO mice with other AD mice model is essential to ascertain whether miR-455-3p can lessen the toxicities of full-length APP in mice (Kumar et al., 2021b). This is especially true for mice with an unmodified 3’ UTR, such as the recently created humanized Ab-KI mice. The subsequently created double mutant animals, hAb-KI X TG miR-455-3p, were projected to be less dangerous to mitochondria and synaptic activity from APP and its c-terminal derivative, as well as to live longer and exhibit less cognitive decline. In contrast, it was projected that mice with double mutant hAb-KI X KO miR-455-3p would live shorter lives, experience more cognitive decline, and develop AD pathologies more quickly (Kumar et al., 2021b; Kumar et al., 2019b).

The main physiological indicator of AD etiology is cognitive dysfunction. Age-matched WT and TG mice fared better on the MWM (Morris Water Maze) test in terms of cognitive learning and memory, but KO mice did poorly. These findings imply that increased amount of endogenous miR-455-3p enhances cognitive abilities (Honer et al., 2019; John and Reddy, 2021; Kennedy, 2016; Kumar and Reddy, 2020). The brains and hippocampal regions of TG mice had noticeably more synapses in addition to greater amounts of the proteins MAP2, PSD95, and SNAP25. Furthermore, the hippocampal neurons of miR-455-3p TG mice exhibited higher dendritic spine density. It is yet unknown how miR-455-3p overexpression upregulates synaptic activity and proteins. It is commonly known that improving synaptic proteins and upregulating PGC1α improves cognitive function (Cheng et al., 2012; Honer et al., 2019). According to recent researches, cognitive performance can be enhanced by upregulating miR-455-3p by inhibiting EphA4 and HDAC2. Increased miR-455-3p levels lessen neurotoxicity by preventing hippocampus neurons from producing EphA4. Subsequent research highlighted the role of miR-455-3p in correcting neurological abnormalities and fixing memory impairments in TBI (traumatic brain injury) patients. It has been discovered that cinnamic acid reduces memory deficits in TBI patients by downregulating HDAC2 and upregulating the miR-455-3p expression levels (Guo et al., 2019; Zhu et al., 2020).

We also found that miR-455-3p directly improved neuronal differentiation. In TG mice, NeuN labelling demonstrated a notable rise in the neuronal population. Increased amount of the neural marker NeuN and dendritic marker MAP2 further supported the enlarged neuronal population caused by miR-455-3p overexpression. It is intriguing to note that the astrocyte population was subsequently lower in TG mice than in KO and WT animals. As expected, KO mice with miR-455-3p knockdown showed decreased neuronal population and increased expressions of astrocytes and microglias (Kumar et al., 2021b). The development of abnormal mitochondrial function and structure is a major source of synaptic dysfunction in AD. miR-455-3p affected the shape and function of mitochondria, according to previous studies (Calkins and Reddy, 2011; Reddy, 2011). The increase in mitochondrial length and number in TG mice demonstrated improved mitochondrial organization, indicating higher mitochondrial quality, according to our TEM analysis. An increase in the quantity of proteins involved in mitochondrial biogenesis enforced the improvement. The TG mice showed positively regulated dynamics proteins (MFN2, MFN1, FIS1, DRP1, and OPA1) and elevated expressions of mitochondrial biogenesis proteins (NRF2, TFAM, NRF1, and PGC1α). However, there are several opinions on how miR-455-3p affects mitochondrial activities. miR-455-3p’s regulation of HIF1α is one of the recognized explanations (Zhang et al., 2015). Moreover, the correlation between HIF1α and PGC1α in controlling mitochondrial activity indicates that miR-455-3p functions as a key mediator of mitochondrial activity in TG mice by way of HIF1α and PGC1α (O’Hagan et al., 2009; Yun et al., 2019).

In conclusion, miR-455-3p improves brain health generally and has a good effect on neuronal activity via a variety of mechanisms. More genetic crossover research employing APP TG animal lines and hAb-KI is required to completely comprehend miR-455-3p’s protective actions against mutant APP proteins. These animals offer significant opportunities for studying the physiological pathways of miR-455-3p in AD and other human disorders.

1.3. miR-455-3p as a potential peripheral biomarker for AD

Based on the above findings and discussion, miR455-3-p emerges as a potential candidate to be considered as biomarker for AD. Here, we discuss on the role of different miRNAs as biomarkers for AD, Reddy lab findings for miR455-3-p as a biomarker, and a scheme for developing miR-455-3p Kit for AD and MCI diagnosis.

a. MicroRNAs role as a biomarker in AD

It has been demonstrated that microRNAs, which are tiny non-coding RNAs, control the expression of certain genes. Changes in miRNA expression have been linked to various pathological processes, including neurodegeneration (Femminella et al., 2015). In the search for easily accessible, non-invasive biomarkers for AD diagnosis and prognosis, circulating miRNAs are among the most promising candidates (Femminella et al., 2015). Several of them have been repeatedly found in AD, and their targets likewise appear to be involved in the pathophysiological mechanisms that underlie AD. Certain miRNAs were expressed in the central nervous system (CNS), where they control neurite outgrowth, synaptic plasticity, and neuronal development. Except for the hippocampus, Landgraf et al. (2007) showed that human adult brain regions could be distinguished from adult rat brain regions using cluster analysis based on the expression of orthologous miRNAs. Additionally, adult and embryonic brain tissues form distinct clusters (Landgraf et al., 2007). Several miRNAs potentially related AD pathology have been consistently identified as dysregulated in AD (Van den Hove et al., 2014). For example, miR-455, miR-191, miR-106, miR-146a, miR-29, miR-34, miR-107, miR-181 etc. One of the most commonly changed miRNAs in AD is miR-9, which is encoded by three distinct genes. It has been demonstrated that adding Aβ peptides to primary neuronal cell cultures would downregulate this tiny RNA, which is abundantly expressed in the prenatal hippocampal region and downregulated in AD brains (Krichevsky et al., 2003). Early in AD, there has been evidence that the short RNA miR-107 is downregulated in the temporal cortex. Its expression has been shown to target BACE1, hence controlling the synthesis of amyloid, and it also has a negative connection with neuritic plaque density and neurofibrillary tangles (Nelson and Wang, 2010).

The miR-29 family of miRNAs has been demonstrated to enhance amyloid formation in vitro neuronal models and exhibit an inverse correlation with BACE1 (Hébert et al., 2008). The study showed downregulation in the cerebellum, temporal cortex, and patient serum of human AD patients (Hébert et al., 2008). On the other hand, miR-34 controls p53 expression in AD, which is linked to tau phosphorylation (Hooper et al., 2007). Moreover, transgenic mice models of AD and the hippocampi of AD patients both exhibit elevated levels of miR-34 (Zovoilis et al., 2011). Whereas miR-181 functions as a gene regulator for oncogene RAS and tumor necrosis factor-alpha. Aβ has the ability to downregulate miR-181 expression in primary human astrocyte cultures, and in vitro tests have demonstrated a correlation between miR-181 and Aβ levels in the human AD brain (Schonrock et al., 2012). It has been demonstrated that miR-106a and miR-106b directly bind to APP mRNA and are downregulated in AD patients’ temporal cortical regions (Kim et al., 2012). In addition to the miRNAs mentioned earlier, others including miR-124, miR-132, miR-153, and miR-146a have also been linked to AD and other neurodegenerative conditions. Some of them appear to be engaged in more than one of these processes, indicating that they may also mediate cross-talk among the various pathogenic processes underlying AD. The majority of them have demonstrated significance in tau phosphorylation, neuroinflammation, APP processing, and ApoE lipidization (Sethi and Lukiw, 2009) (Goodall et al., 2013; Van den Hove et al., 2014).

Mild cognitive impairment (MCI) is characterized as a diverse clinical syndrome marked by notable changes in cognitive function and deficits identified through neuropsychological testing, while overall daily functionality remains relatively intact (Winblad et al., 2004). The diagnostic criteria for MCI have evolved over time (Ogonowski et al., 2022). Finding novel biomarkers for the diagnosis and prognosis of MCI is a major task. Within this context, serum, plasma, and other bodily fluids’ miRNAs have become a potentially useful source of biomarkers for cognitive deficits associated with MCI and AD. It is interesting to note that, in response to pathophysiological stimuli, miRNAs can control various signaling pathways via a variety of targets (Ogonowski et al., 2022). The “miR-134 family” (miR-134/miR-370, miR-323-3p/miR-370, and miR-382/miR-370) as well as the “miR-132 family” (miR-128/miR-491-5p, miR-132/miR-491-5p, and mir-874/miR-491-5p) comprise the identified biomarker pairs (Sheinerman et al., 2013). miRNAs such as miR-27b-3p, miR-92a-3p, miR-181c-5p, miR-181a-5p, miR-210-3p, and miR-455-3p that have been connected to AD and MCI, have been demonstrated to be elevated, whereas miR-93-5p, miR-107, miR-127-3p, miR-142-3p, and miR-363-3p, miR-15b-3p, miR-22-5p, miR-29b-3p, miR-30c-5p, and miR-30b-5p were among the 67% of miRNAs that were downregulated. This finding raises the possibility that these 16 miRNAs are predictive biomarkers for MCI (Ogonowski et al., 2022). Considering these, the role of miRNAs as biomarkers in AD and MCI has been a rapidly growing area of research. Their ability to reflect underlying molecular changes, their presence in accessible biofluids, and their dynamic expression profiles make them valuable tools for early diagnosis, prognosis, and therapeutic monitoring. As research advances, the integration of miRNA biomarkers into clinical practice holds the promise of improved management and treatment of AD and MCI, ultimately enhancing patient outcomes.

b. miR-455-3p as a biomarker for AD

The Reddy lab has discovered miR-455-3p as a viable peripheral biomarker for AD (Kumar and Reddy, 2019). We (Reddy lab) measured miRNAs in serum samples from persons with MCI and AD in comparison to healthy controls in order to accomplish our goal. Affymetrix microarray analysis was utilized, and qRT-PCR was employed to confirm the differentially expressed miRNAs. Furthermore, utilizing AD cell lines, AD fibroblasts, AD cerebrospinal fluids, transgenic mice for the amyloid precursor protein, AD B-lymphocytes (all from late-onset AD; LOAD), and postmortem brains from AD patients, we found that AD cases had higher expression levels of miR-455-3p than samples from healthy controls. We further confirmed the miRNA results. We observed a progressive increase in the expression of four miRNAs: miR-455-3p, miR-4668-5p, miR-3613-3p, and miR-4674. When compared to controls, those with AD and mild cognitive impairment had lower levels of mir-6722. Among them, qRT-PCR validation analysis revealed that only miR-455-3p (P = 0.007) and miR-4668-5p (P = 0.016) were significantly upregulated in AD patients as compared to healthy controls. When compared to controls, it revealed an astonishing 11.3-fold increase in expression for miR-455-3p in AD patients. Additionally, miR-455-3p was shown to be upregulated (P = 0.016) in the AD postmortem brains with varying Braak stages according to qRT-PCR analysis. Nevertheless, A significant area under the curve (AUC) value was only found for miR-455-3p in the serum (AUROC = 0.79; P = 0.015) and brains (AUROC = 0.86; P = 0.016) of AD patients, according to receiver operating characteristic curves (ROC) curve analysis. Again, the cerebral cortex, the region of the brain affected by AD, had a 1.8-fold high level of miR-455-3p (P = 0.004), while the cerebellum, the non-affected area, had a low level of this protein, according to an expression study of transgenic mice for amyloid precursor protein. Moreover, miR-455-3p was equally overexpressed in the Aβ (1–42) peptide treatment of neuroblastoma cells in humans and mice. The Aβ-treated SH-SY5Y cells exhibited a 4.1-fold (P = 0.027) increase in hsa-miR-455-3p expression in comparison to the control (untreated) cells (Figure 6). Likewise, in N2a cells, the expression of miR-455-3p was elevated by 3.8-fold (P = 0.021) in Aβ-treated cells as opposed to control cells. Functional investigation using the miR-path on differentially expressed miRNAs revealed that miR-455-3p was involved in the control of multiple biological pathways.

Figure 6:

This image illustrates the elevated expression levels of miR-455-3p in AD related specimes; serum and CSF collected from AD and MCI patients, postmortem AD brain sections, and AD cell culture models.

It was discovered that genes connected to these pathways play a critical role in the pathophysiology of AD. Several signaling networks and a number of AD-related genes have been directly connected to the pathophysiology of the disease. The link between miR-455-3p and 11 biological pathways and related genes was determined by the miRNA analysis. The TGF-β signaling pathway, the hippo signaling pathway, the ECM-receptor interaction, and the control of the actin cytoskeleton were the most significant signaling pathways (Kumar et al., 2017b). Another study from Reddy’s lab (Kumar and Reddy, 2018b) reported that, compared to healthy control subjects, fibroblasts and B-lymphocytes from AD patients had higher levels of miR-455-3p. Notably, sporadic AD patients’ cells had much higher levels of miR-455-3p than did the healthy control’s cells. In particular, B-lymphocytes from sporadic AD individuals had considerably greater levels of miR-455-3p controls (P = 0.044). An examination of the Receiver Operating Characteristic (ROC) curve showed that AD fibroblast cells (AUROC = 0.861; P = 0.03) and postmortem brains (AUROC = 0.792; P = 0.001) had a substantial area under the curve (AUROC) value for miR-455-3p. These results clearly suggest that miR-455-3p is a potential biomarker for AD (Kumar and Reddy, 2018b). However, more research is required to elucidate the molecular connections between miR-455-3p and the progression of AD. From these findings, we can infer that miR-455-3p might serve as a potential peripheral biomarker for diagnosing AD and MCI. While numerous studies have suggested miRNAs as potential markers for AD, no one miRNA has yet to be clinically verified as a reliable biomarker. The reported findings assist in identifying a promising candidate, with the expression levels of miR-455-3p suggesting its potential role as a peripheral biomarker (Kumar et al., 2017b).

c. Development of a miR-455-3p kit for MCI and AD diagnosis

Based on the above findings and discussions (Kumar and Reddy, 2018b; Kumar et al., 2017b), Dr. Reddy’s lab is developing a biochemical kit utilizing miR-455-3p for the diagnosis of AD and MCI. By developing this kit, we can detect MCI and AD in patients early, allowing for timely therapeutic interventions that can potentially slow the progression of these conditions, thereby improving patient outcomes and quality of life. Additionally, miR-455-3p can be measured in blood samples, making the diagnostic test minimally invasive compared to procedures like cerebrospinal fluid analysis or brain imaging. A blood-based test is more comfortable and less risky for patients, particularly the elderly, who may find other diagnostic procedures invasive and stressful. Focusing on a specific microRNA consistently altered in AD and MCI, the kit can provide a more sensitive and reliable diagnostic tool, reducing the chances of false positives and negatives. Early and accurate diagnosis of MCI and AD can also reduce healthcare costs by preventing or delaying the need for more intensive care and treatment as the disease progresses. Moreover, with a reliable diagnostic tool, healthcare resources can be better allocated to those who need them most, improving overall healthcare efficiency and patient care. In summary, the development of a miR-455-3p diagnostic kit is crucial for improving the early detection, diagnosis, and management of MCI and AD. It holds promise for enhancing patient outcomes and optimizing healthcare resources.

However, the results obtained from the miR-455-3p diagnostic kit should be corroborated with additional assessments to confirm the disease, including brain scans and the Montreal Cognitive Assessment (MoCA). The official scoring range for the MoCA is as follows: normal cognition - 26–30 points, mild cognitive impairment - 18–25 points, moderate cognitive impairment - 10–17 points, and severe cognitive impairment - under 10 points. Additionally, it is important to test for cell-free DNA and DNA double-strand breakage to get a comprehensive diagnostic profile.

For the development of the miR-455-3p diagnostic kit, Dr. Reddy’s lab proposes the following steps:

1. Blood Collection: Collect blood samples from patients.

2. Serum Preparation: Separate the serum from the blood and store it at the appropriate temperature.

3. RNA Isolation and cDNA Synthesis: Isolate total RNA from the serum, perform polyadenylation, and synthesize cDNA using miR-455-3p specific primers.

4. Gene Expression Analysis: Normalize and quantify gene expression. Calculate miR-455-3p fold changes using quantitative reverse transcription–polymerase chain reaction (RTqPCR). (Figure: 6).

Additionally, the miR-455-3p levels will be validated using fluorescence spectra, following the procedures described by Guo and colleagues (Guo et al., 2022).

It is anticipated that the fluorescence spectral analysis of miR-455-3p will authenticate the miR-455-3p data and increase the accuracy of AD diagnosis. In this process, a dual-signal DNA probe based on the fluorescence recovery of carbon quantum dots (CQDs) and the sulfo-cyanine5 (Cy5) dye will be used to determine miR-455-3p simultaneously. In order to amplify the signals through a cycle of DNA degradation and RNA re-hybridization, duplex-specific nuclease will be added. The method’s dynamic ranges were 0.01 to 4 pM for miR-455-3p and 0.01 to 5 pM for that same compound. The recovery of miRNAs in spiking human serum and its strong correlation with RT-qPCR testing will validate the method’s accuracy. All enrolled individuals, including healthy donors, MCI patients, and AD patients, will have their blood samples tested to measure the miRNA content. However, a straightforward and sensitive technique for the very accurate simultaneous measurement of miRNAs has been published, and it suggests that the concentration ratio of miR-455-3p is a useful blood indicator for the early and in-stage diagnosis of AD (Guo et al., 2022).

Conclusions and Future Directions

An ideal biomarker for diagnosing MCI and AD progression should be early detectable, non-invasive in nature, preferably peripherally available. We reported a significant upregulation of mir-455-3-p expression in AD postmortem brains, AD fibroblasts, and B-lymphocytes. We also found high level of mir-455-3-p in AD serum, which was later validated by the presence of an increased miR-455-3p in CSF exosomes secreted from the brain. miR-455-3p targets many genes related to oxidative decay, mitochondrial dysfunction, synaptic damage and neurodegeneration process in AD pathogenesis. More importantly, miR-455-3p dramatically decreases full-length APP expression, the C-terminal fragment of APP and reduces Aβ(1–40) and Aβ(1–42) levels. Our in-vitro cell culture studies strongly explains the role of miR-455-3p in protecting neuronal cells against AD related stress including mutant APP. Moreover, mir-455-3-p mice models showed its vibrant role in maintaining and promoting memory and cognitive functions in an extended life period. Overall, its positive role in the regulation of mitochondrial biogenesis/dynamics and synaptic activity make it worthy of research to identify and validate AD diagnosis and our proposed miR-455-3p detection kit will serve the purpose largely.

Figure 7:

Diagram showing the progression of the miR-455-3p detection kit development: Twenty milliliters of blood will be drawn, and it will be processed right away to separate the buffy coat and serum components. Following the isolation of total RNA, a set of procedures will be carried out, including polyadenylation and the synthesis of cDNA using primers specific to miR-455-3p. The study of fluorescence spectra will be used to confirm the correctness of miR-455-3p levels.

Highlights.

• MicroRNAs are small non-coding RNAs evolutionary conserved molecules that are involved with Alzheimer’s disease.

• MicroRNAs regulate cellular processes, including RNA silencing, post-translational gene expression and neurodegeneration.

• Overexpression of miR-455-3p in mice showed superior cognitive learning, improved memory and extended lifespan.

• Reddy lab research revealed that microRNA-455-3P is a potential biomarker and therapeutic target for AD.

Abbreviations:

ACVR2B

Activin Receptor Type IIB

AD

Alzheimer’s disease

ADRD

Alzheimer’s disease related disease

AGO

Argonaute

AMPK α1

AMP-activated kinase α1 subunit

APHA1

Aph-1 Homolog A

APOE4

apolipoprotein E4

APP

Amyloid precursor protein

ARE

Antioxidant response elements

Aβ

beta-amyloid

BACE1

Beta-site APP cleaving enzyme-1

BMP4

Bone morphogenetic protein 4

CD2AP

CD2 Associated Protein

CHRDL1

Chordin-like 1

CNS

Central nervous system

COL27A1

collagen type XXVII alpha 1 chain

CQDs

Carbon quantum dots

CSF

Cerebrospinal fluid

Cy5

Cyanine5

DGCR8

DiGeorge Syndrome Critical Region 8

DLD

Dihydrolipoamide dehydrogenase

DNA

Deoxyribonucleic acid

EPHA4

Ephrin Type-A Receptor 4

HDAC2

Histone deacetylases 2

HIF1an

hypoxia-inducible factor 1 α inhibitor

Hmgb1

High-mobility group box

IPF

Idiopathic pulmonary fibrosis

KO

Knockout

MCI

Mild cognitive impairment

MEKK1

Mitogen-activated protein kinase kinase 1

mir-455-3-p

MicroRNA-455-3-p

miRISC

miRNA-induced silencing complex

MoCA

Montreal cognitive assessment

mRNA

Messenger RNA

MS4A4

membrane-spanning 4-domains subfamily A

ncRNA

Noncoding RNAs

NFTs

Neurofibrillary tangles

NGF

Nerve growth factor

NRF2

nuclear factor erythroid 2-related factor 2

OGD

Oxygen and glucose deprivation

PAK2

P21-activated kinase 2

PDCD7

Programmed cell death 7

pre-miRNAs

precursor-miRNAs

pri-miRNAs

primary miRNAs

PS1

presenilin 1

PS2

presenilin 2

PTEN

Phosphatase and tensin homolog

RISC

RNA-induced silencing complex

RNA

Ribonucleic acids

ROC

Receiver operating characteristic

RT-qPCR

Quantitative reverse transcription–polymerase chain reaction

SNHG15

Small nucleolar RNA host gene 15

SOX2-OT

Sex-determining region Y-box 2 overlapping transcript

TBI

Traumatic brain injury

TG

Transgenic

TGF-β

Transforming growth factor beta

TP53INP1

Tumor protein p53 inducible nuclear protein 1

TRAF3

TNF Receptor Associated Factor 3

TRAF6

Tumor necrosis factor receptor associated factor 6

TβRI

TGF-β type I receptor

TβRII

TGF-β type II receptors

UTR

Untranslated region

ZEB1

Zinc finger E-box-binding homeobox 1