Regulation of neurotropic signaling by the inducible, NF-kB-sensitive miRNA-125b in Alzheimer's disease (AD) and in primary human neuronal-glial (HNG) cells

Abstract

Inducible microRNAs (miRNAs) perform critical regulatory roles in central nervous system (CNS) development, aging, health, and disease. Using miRNA arrays, RNA sequencing, enhanced Northern dot blot hybridization technologies, Western immunoblot, and bioinformatics analysis, we have studied miRNA abundance and complexity in Alzheimer's disease (AD) brain tissues compared to age-matched controls. In both short post-mortem AD and in stressed primary human neuronal-glial (HNG) cells, we observe a consistent up-regulation of several brain-enriched miRNAs that are under transcriptional control by the pro-inflammatory transcription factor NF-kB. These include miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155. Of the inducible miRNAs in this subfamily, miRNA-125b is among the most abundant and significantly induced miRNA species in human brain cells and tissues. Bioinformatics analysis indicated that an up-regulated miRNA-125b could potentially target the 3'untranslated region (3'-UTR) of the messenger RNA (mRNA) encoding (a) a 15-lipoxygenase (15-LOX; ALOX15; chr 17p13.3), utilized in the conversion of docosahexaneoic acid into neuroprotectin D1 (NPD1), and (b) the vitamin D3 receptor (VDR; VD3R; chr12q13.11) of the nuclear hormone receptor superfamily. 15-LOX and VDR are key neuromolecular factors essential in lipid-mediated signaling, neurotrophic support, defense against reactive oxygen and nitrogen species (reactive oxygen and nitrogen species), and neuroprotection in the CNS. Pathogenic effects appear to be mediated via specific interaction of miRNA-125b with the 3'-UTR region of the 15-LOX and VDR messenger RNAs (mRNAs). In AD hippocampal CA1 and in stressed HNG cells, 15-LOX and VDR down-regulation and a deficiency in neurotrophic support may therefore be explained by the actions of a single inducible, pro-inflammatory miRNA-125b. We will review the recent data on the pathogenic actions of this up-regulated miRNA-125b in AD and discuss potential therapeutic approaches using either anti-NF-kB or anti-miRNA-125b strategies. These may be of clinical relevance in the restoration of 15-LOX and VDR expression back to control levels and the re-establishment of homeostatic neurotrophic signaling in the CNS.

Figure 1. Up-regulated AD-relevant miRNAs regulated by NF-kB

primary HNG cells were treated with an AD-relevant, NF-kB-inducing cocktail of Aβ42 peptide (5 M; Sigma-Aldrich) plus human recombinant interleukin-1β (IL-1β; 10 nM; I4019, Sigma-Aldrich Chemical, St. Louis, MO); Aβ42 peptides were prepared using the hexafluoroisopropanol (HFIP) evaporation-dimethyl sulfoxide-solubilization method as previously described [10,39,40]. Confirmation of selective miRNA induction and NF-kB sensitivity was obtained using (a) RNA sequencing; (b) LED-Northern dot blot and/or (c) RT-PCR analysis [10,39]; (d) by inhibition of this induction using specific NF-kB inhibitors CAPE, CAY10512 and PDTC [10,39,64] and (e) by bioinformatics analysis of functional NF-kB binding sites in the promoters of the genes that encode these inducible miRNAs [10,39,60]. A small family of 5 miRNAs including miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155 were found to be up-regulated in both stressed HNG cells and in high quality total RNA isolated from short post-mortem AD brain; note that hsamiRNA-128 is an example of a variably up-regulated miRNA; miRNA-183 is an internal control miRNA that is neither up- nor down-regulated after induced stress; N=5; other up-regulated miRNAs showed modest up-regulation but are not discussed further here [10,39,64].

Figure 2. Complementarity maps between homo sapiens VDR or 15-LOX mRNA 3′-UTRs and miRNA-125b

[A] schematic representation of human VDR mRNA, highlighting a section (black box) of the 3′-untranlated region (3′-UTR) and the complementarity of this VDR mRNA 3′-UTR region (red) with the predicted target sequence of homo sapien (hsa) miRNA-125b (yellow); size and scale are approximate; the human VDR mRNA (encoded from a 9 exon gene at chr12q13.11; GenBank: AB002168.2)contains about 4669 nucleotides followed by a variable poly A tail [50; http://www.Genecards.org/cgi-bin/carddisp.pl?gene=VDR; Weizmann Institute of Science, Rehovot, Israel]; meCAP = 5′ methyl cap typical of all eukaryotic mRNAs and ORF = open reading frame; the numbering refers to the 5′ end of the mRNA as 1; a ‘:’ indicates a partial hydrogen bond and an ‘I; indicates a perfectly complementary hydrogen bond; ribonucleotides in upper case are implicated in either partial or full hydrogen bonding; [B] is a schematic representation of human 15-LOX (ALOX15; arachidonate 15-lipoxygenase) mRNA, highlighting a section of the 3′-UTR (black box) and the complementarity of this 15-LOX mRNA 3′-UTR region (red) with the predicted target sequence of hsa miRNA-125b (yellow); the hsa 15-LOX mRNA (encoded by a 14 exon gene at chr 17p13.3; GenBank: U88317.1) is considerably smaller than the hsa VDR mRNA; size and scale are approximate; the human 15-LOX mRNA contains about 1707 nucleotides followed by a variable poly A tail [see http://www.genecards.org/cgi-bin/carddisp.pl?gene=ALOX15&search=15-lox.

Figure 3. As in AD brain, 15-LOX and VDR expression is down-regulated in stressed HNG cells

Western analysis; HNG cells were cultured as previously described in detail [10,40,47,67,72]. An AD-relevant stress cocktail of [Aβ42+IL-1β] together was found to reduce 15-LOX [A] and VDR [B] protein levels to approximately 0.25- and 0.35-fold, respectively, of their control, homeostatic levels in these HNG cell co-cultures [10,40,67]; human antibodies for 15-LOX and VDR were respectively [(C-20): sc-1008] and [(H235); sc-32940; Santa Cruz Biotechnology, Santa Cruz CA; www.scbt.com); Western analysis yielded 2 bands for both 15-LOX (~80 and ~75 kDa) and VDR (~48 and ~56 kDa); both bands were quantitated for the bar graph in [C]; the reductions in both 15-LOX and VDR in stressed HNG cells compared to untreated control are highly significant compared to an unchanging β-actin signal in the same sample; N=4, *p<0.01 (ANOVA); a dashed horizontal line at 1.0 indicates control, homeostatic levels for 15-LOX (left panel) and VDR (right panel) for ease of comparison.

Figure 4

Taken together, recent findings define in part a highly interactive network of NF-kB-sensitive, up-regulated miRNAs in stressed human brain cells and AD brain that can explain much of the observed neuropathology in AD. The CNS-abundant, miRNA-125b is a central member of this up-regulated miRNA group that may be in part responsible for driving deficits in phagocytosis (TREM2), innate-immune signaling and chronic inflammation (IkBKG, CFH), impairments in neurotransmitter packaging and release (SYN-2), and neurotrophism (15-LOX, VDR). Other NF-kB-sensitive up-regulated miRNAs (such as miRNA-146a) appear to be responsible for the observed deficits in NF-kB regulation (IRAK-1, IRAK-2) and/or amyloidogenesis (TSPAN12) (see text); these up-regulated miRNAs and down-regulated mRNAs form a highly integrated, pathogenic miRNA-mRNA signaling network resulting in gene expression deficits in sporadic AD that may be self-perpetuating due to chronic re-activation of NF-kB stimulation perhaps through the involvement of deficits in IkBKG signaling [10,73-76]. Inhibition of the NF-kB initiator or individual blocking of the pathogenic induction of these 5 miRNAs may provide novel therapeutic approaches for the clinical management of AD, however what NF-kB or miRNA inhibition strategies, or whether they can be utilized either alone or in combination, remain open to experimentation (see Figure 4). Extensive recent data in human brain cells in primary culture has indicated that these approaches may neutralize this chronic, inducible, progressive pathogenic gene expression program to re-establish brain cell homeostasis, and ultimately be of novel pharmacological use in the clinical management of AD.

Figure 5. Western analysis; recovery of 15-LOX and VDR expression using anti-miRNA-125b or anti-NF-kB treatment strategies in vitro

AD-relevant stress (a cocktail of Aβ42+IL-1β; see Figure 1) reduces 15-LOX and VDR protein levels to respectively 0.25- and 0.35-fold of their control, homeostatic levels in primary HNG cell co-cultures [10,40,67]; while a control scrambled anti-miRNA-125b (AM125b.sc) has no effect on restoring protein levels (black bars), a fully antisense, protected anti-miRNA-125b (AM125b; light gray bars) restores expression levels to near control levels; for ease of comparison a dashed horizontal line at 1.0 indicates pre-stress (control) levels for 15-LOX and VDR; similarly the NF-kB inhibitors pyrollidine dithiocarbamate (PDTC; medium gray bars) and the polyphenolic resveratrol analog trans-3,5,4′-trihydroxy-stilbene CAY10512 (dark gray bars) [10,39,40] show near equal restoration of 15-LOX and VDR signals back to homeostatic (control) levels; these data and recently published data indicate that anti-miRNA (AM, antagomir) and/or anti-NF-kB therapeutic strategies may be useful in restoring homeostatic gene expression in AD; N=3; *p<0.01 (ANOVA); see also text and Figures 1 and 3.