A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

Abstract

Neurodegeneration associated with amyloid β (Aβ) peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD). Numerous microRNAs regulate amyloid precursor protein (APP) expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3′ untranslated region (UTR) of the mRNA encoding Ran-binding protein 9 (RanBP9). RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1), secretion of soluble APPβ (sAPPβ), and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3′-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs), located downstream of the enhanced green fluorescence protein (EGFP) open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on the pathology of AD.

Targeting the HDAC2/HNF-4A/miR-101b/AMPK Pathway Rescues Tauopathy and Dendritic Abnormalities in Alzheimer’s Disease

“…32 Notably, miR-101 is shown to downregulate APP and fibrillar Ab in hippocampal neurons by directly targeting APP 33 or RanBP9, which encodes for the RanBP9 protein to enhance the proteolytic processing of APP and Ab generation both in vivo and in vitro. 34 In the present study, we demonstrate that miR-101b directly targets the 3 0 UTR of AMPK to post-transcriptionally regulate its protein levels. We further identified a physical binding of the HNF-4A transcription factor to the promoter region of miR-101b.…”

Noncoding RNAs in Alzheimer's disease

“…Additional studies are necessary to understand the complex network of regulatory microRNAs that suppress BACE1 production in order to consider their potential use in AD therapy. (Nelson & Wang, 2010;Wang et al, 2008) miR-29a/b-1 BACE1, palmitoyltransferase (Hébert et al, 2008) miR-9 BACE1 (Hébert et al, 2008) miR-29c BACE1 miR-188-3p BACE1 miR-339-5p BACE1 (Long et al, 2014) miR-195 BACE1, Tau (Ai et al, 2013;Sun et al, 2015;Zhu et al, 2012) MiR-186 BACE1 miR-27a-3p Presenilin-1 (Sala Frigerio et al, 2013) miRNA-34a Tau, γ-secretase (Dickson et al, 2013;Jian et al, 2017) miRs-24, miR-186, miR-455 Nicastrin (Delay et al, 2014) miR-107 ADAM10 (Augustin et al, 2012) miR-144 ADAM10 miR-125b, miR-146a Tetraspanin12 Xu et al, 2009) miR-101 APP (Barbato et al, 2014;Vilardo et al, 2010) miR-16 APP (Liu et al, 2012;Zhang et al, 2015) miR-147, miR-20a APP miR-137, miR-181c, miR-9, miR-29a/b palmitoyltransferase (Geekiyanage & Chan, 2011;Schonrock et al, 2012) miR-124 PTBP1, BACE1 (Fang et al, 2012;Smith et al, 2011) miR-132, miR-212 GSK3, Tau (Smith et al, 2015) miR-125b, miR-138, miR-106b, miR-922 Tau phosphorylation (Banzhaf-Strathmann et al, 2014; miR-15, miR-16 ERK1 (Hébert et al, 2010) miR-33 ABCA1 and APOE lipidation (Kim et al, 2015) piRNAs piR-38240 KPNA6 (Roy et al, 2017) aca-piR-4, aca-piR-15 CREB1 (Rajasethupathy et al, 2012) lncRNAs BACE1-AS BACE1 (Faghihi et al, 2008;Kang et al, 2014;Liu et al, 2014) lncRNA 51A SORL1 (Ciarlo et al, 2013) BDNF-AS (NAT) BDNF (Modarresi et al, 2012) LRP1-AS LRP1 (von Einem et al, 2010;…”

“…Several microRNAs have been shown to directly reduce APP biosynthesis and thus the generation of Aβ plaques (Basavaraju & de Lencastre, 2016). For example, miR-101 targeted APP mRNA and hence suppressed APP production, cleavage and accumulation of fibrillar Aβ; conversely, inhibition of miR-101 increased full-length APP (Barbato et al, 2014;Vilardo, Barbato, Ciotti, Cogoni, & Ruberti, 2010). miR-16 was also found to target APP mRNA and suppressed APP levels, and low miR-16 induced APP accumulation in AD mice (Liu et al, 2012).…”

Sponging of cellular proteins by viral RNAs

“…The introduction of designer RNA sponges has been demonstrated to be an effective means of down-regulating the functions of major miRNAs such as miR23b [10], miR-101 [11], miR-151 [12] and others [13]. These synthetic RNA sponges have been delivered using a variety of vectors – including lentiviruses.…”