In the present study, we systematically identified a potential PBMC-based miRNA biomarker for DAT. Our initial miRNA profiling allowed the identification of miRNAs with high differential expression in DAT and MCI individuals compared to HS individuals which was consistently upregulated in the discovery cohort and independent cohorts. Expression levels of miR-150-5p was upregulated in PBMC of patients with DAT and was closely correlated to cognitive performance, CSF Aβ1−42 and tau levels, as well as MRI AD pattern of atrophy. Our results demonstrated that higher levels of miR-150-5p was associated with derogatory effects on brain structure, especially in regions of the brain belonging to the default mode and executive control networks, key brain networks implicated in AD. Furthermore, miR-150-5p was subjected to miRNA targets prediction, functional enrichment analysis, and verification of miRNA targets expression. Our bioinformatics analysis revealed that selected targets of miR-150-5p were markedly associated with Wnt signaling pathway which has been implicated in AD [36]. We further showed that target genes PDCD4 expression levels were downregulated in DAT. These results provide evidence that upregulation of miR-150-5p suppresses its downstream target genes. Therefore, our findings suggest that miR-150-5p is a potential as a biomarker for the diagnosis of DAT.
A previous study showed that PBMC is involved in the pathogenesis of AD due to its central role in inflammation during the early phase of AD development [37]. Lymphocytes and monocytes, which represent the majority of immune cells in PBMC, can infiltrate blood brain barrier (BBB) and lead to inflammatory cascade in the brain of AD [38]. Furthermore, it is also reported that dysfunction of BBB in AD enables exchange of miRNAs between the brain and peripheral blood [39]. A recent review reported that a higher correlation of transcriptomic changes (pattern of gene expression) and epigenomic changes (pattern of DNA methylation) in the CNS and blood [40]. Taken together, it is hypothesized that that changes in the expression of miRNAs in the PBMC could reflect the neuropathogenic changes in the CNS and PBMC may serve as a potential source to find miRNAs signatures that could help to discriminate cognitively healthy subjects from diseased patients. To explore the role of PBMC derived miRNAs for DAT, we unbiasedly profiled the miRNAs from PBMC of 54 study participants. (Fig. 1a). Additionally, we validated the aberrantly expressed miRNA in an independent cohort of 64 study participants. As verified by qPCR, miR-150-5p was upregulated in DAT versus HS individuals and DAT versus MCI individuals with an AUC of 0.86 and 0.86, respectively (Fig. 2a and b).
In addition to differential expression of miRNA in HS, MCI, and DAT individuals, another important consideration is the reliability of the miRNA with regards to cognitive, neuroimaging, and CSF biomarkers for AD. In this regard, we observed miR-150-5p expression levels to be inversely correlated to cognitive tests including MMSE and MoCA, demonstrating that higher miR-150-5p levels was associated with lower cognitive scores (higher disease severity) (Fig. 3a and b). Our findings also demonstrate that miR-150-5p expression levels were negatively correlated with CSF Aβ1−42 levels and positively correlated with CSF tau levels (Fig. 3c and d). In addition, subjects with DAT also had lower CSF Aβ1−42 and higher CSF tau compared to HS and MCI subjects. As expected, subjects with MCI had intermediate levels of both markers (Table 2). These findings further support the concept that CSF is the gold standard of AD diagnosis where cognitive impaired AD patients with a low concentration of CSF Aβ1−42 are usually accompanied by a higher concentration of CSF tau levels [41]. Furthermore, the concentration of CSF Aβ1−42 in DAT was significantly higher compared to MCI subjects but was comparable to MCI subjects. Similar finding was found in the expression of miR-150-5p among three groups. Thus, not only miR-150-5p expression differentiates DAT from HS, but it also correlates with cognitive performance and CSF Aβ1−42 as well as CSF tau levels and thus has wide clinical implications.
In order to validate miRNAs as biomarkers of AD, there is a need to understand their relationship with in vivo markers of brain structure and AD neuropathology such as amyloid-β and tau. However, there is currently limited evidence illustrating the association between miRNA levels and brain structure in AD. Thus, how miRNA levels influence and relate to in vivo measures of brain structure and function in AD remain a key gap. In this regard, our findings illustrate a derogatory influence of miR-150-5p levels on both voxel-wise and regional grey matter volume in DAT (Fig. 4). Prior studies suggest that effects of miRNA levels on neuropathological markers of AD including amyloid-β and phosphorylated tau could be related to such changes in brain structure. For instance, one study illustrated high accuracy in the diagnosis of prodromal AD using PBMC miRNA markers in accordance with amyloid-β deposition as observed on PET imaging [42]. Evidence also indicates that miRNAs regulate genes that are responsible for amyloid-β and phosphorylated tau up-regulation with increasing evidence suggesting that miRNAs influence amyloid-β production [15]. Additionally, prior studies show alterations of miRNA levels primarily in the grey matter such as those involving the temporal lobes of the brain which are also key regions of amyloid-β and phosphorylated tau deposition in AD [43–45]. Studies in post-mortem human tissue illustrate miRNA expression correlates with the presence of early AD-related pathology in grey matter, specifically with the density of diffuse amyloid-β plaques [45]. Such findings support the notion that patterns of miRNA expression in the cortical grey matter may contribute to, and explain underlying AD pathogenesis [45, 46].
The presence of amyloid-β plaques likely further initiate or potentiate downstream changes that culminate in neurofibrillary tau pathology, cell death, synapse elimination, and eventually cognitive impairment in individuals with altered miRNA levels [45]. Expression of various miRNAs can thus form important candidates in understanding disease progression and modulation in AD. Such events as well as miRNA influence on synaptic loss and synaptic damage are possible mechanisms underlying grey matter loss especially within the hippocampus as well as cognitive decline related to AD. While our group is one of the first to show associations between brain changes and miRNA levels, nonetheless, much remains to be explored regarding the relationship of miRNA levels and brain structure in regions known to be affected in AD.
A single miRNA is able to regulate multiple target mRNAs, and likewise a single target mRNA can be regulated by multiple miRNAs [47]. Using both target prediction and pathway enrichment approaches, we found out some miR-150-5p targets are enriched in the Wnt signaling, PI3K-AKT and thyroid hormone signaling pathways (Fig. 5b). Interestingly, these signaling pathways has been implicated in neurodevelopment and pathogenesis of AD [36, 48, 49]. In addition, miR-150-5p targets were found enriched in the nucleus, nucleoplasm, nuclear membrane, cytosol, cytoplasmic mRNA processing body, and cytoplasm (Fig. 5c), which further demonstrated that the likelihood of miRNA targets shuttles between cytoplasm and nucleus.
It is noteworthy that miRNA negatively regulates the expression of its targets [5, 6]. Consistently, miR-150-5p targets, PDCD4 were found to be downregulated in DAT compared to MCI and HS (Fig. 5d), which reflected the most enriched category for GO biological process that miR-150-5p is a negative regulator (Fig. 5a). PDCD4 (programmed cell death 4), has been demonstrated to be an important tumour suppressor in various cancers by inducing cell apoptosis. Until recently, PDCD4 was found to be regulated by miR-21 via PI3K/AKT signaling pathway in in vitro model of AD [34]. In the treatment of SH-SY5Y cells with Aβ1−42, PDCD4 expression were repressed by miR-21. In this study, our results validated that PDCD4 was remarkably reduced in DAT individuals. Through luciferase assay, we identified that miR-150-5p suppressed expression of PDCD4 at the transcription level. So far, little is known about the biological role of PDCD4 in miR-150-5p-based regulatory mechanisms of AD. Further investigation is warranted.