AbstractBackgroundSmall non‐coding microRNA (miRNA) play a vital role in regulating various biological and pathological processes, including Alzheimer’s disease (AD), brain injury, and head trauma. AD hallmarks include brain amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are the abnormal aggregation of amyloid‐β peptides (Aβ) derived from Aβ precursor protein (APP), and NFTs consist mostly of hyper‐phosphorylated tau proteins. We have recently shown that miRNAs, such as miR‐20b, miR‐153, and miR‐298, can independently regulate AD‐related protein expression (Wang et al‐Mol Psychiatry‐2022). APOE ε4 increases the risk of AD; however, the role of brain miR101 levels in APOE’s influence is presently unknown. Here, we studied the role of miR‐101‐3p (miR‐101) in AD risk and in regulating levels of critical proteins such as APP, GSK3β, and ECE1 implicated in AD.MethodTemporal lobes (TL), cerebellum (CB), and posterior cingulate cortices (PCC) were obtained from non‐cognitively impaired (NCI) and AD subjects, and quantitative RT‐PCR measured miR‐101 levels. The target specificity of miR‐101 was tested using dual reporter clones expressing the 3'‐UTRs of either APP, ECE1, or GSK3β mRNAs. Human microglia and differentiated neuroblastoma (SKNSH) cells were transfected with miR‐101 for 72 hours, and cellular RNA, total proteins, and secreted proteins were analyzed by ELISA and Westerns to understand miR‐101‐mediated regulation.ResultTL and CB regions showed that increased miR‐101 levels were associated with reduced AD risk in the absence of the APOEε4 allele. Interestingly, miR‐101 may increase AD risk in the presence of two APOEε4 alleles. Transfection of miR‐101 altered the translation of several AD‐related proteins, including aggregation‐prone proteins, processing enzymes, and cytokines. Mechanistically, miR‐101 treatment reduced ECE1 and GSK3β mRNA 3’‐UTR activities in reporter clones and protein levels.ConclusionWe have shown that miR‐101 levels modify AD risk in an APOE allele‐dependent manner. Notably, miR‐101 targets the 3’‐UTRs of ECE1 and GSK3β and reduces their expression, making it a key regulator of AD‐related proteins across species and cell types. Our combined results suggest that miR‐101 is critical in regulating essential proteins relevant to AD pathogenesis and could potentially be a novel “multi‐hit” drug target for AD and other neurodegenerative diseases. NIH grants to DKL and SEC.
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