Expression of the phagocytosis-essential protein TREM2 is down-regulated by an aluminum-induced miRNA-34a in a murine microglial cell line

Abstract

One of the key classical pathological features of Alzheimer's disease (AD) is the progressive accumulation of amyloid beta (Aβ42) peptides and their coalescence into highly insoluble senile plaque cores. A major factor driving Aβ42 peptide accumulation is the inability of brain cells to effectively clear excessive amounts of Aβ42 via phagocytosis. The trans-membrane spanning, sensor-receptor known as the "triggering receptor expressed in myeloid cells 2" (TREM2; chr6p21) is essential in the sensing, recognition, phagocytosis and clearance of noxious cellular debris from brain cells, including neurotoxic Aβ42 peptides. Recently, mutations in the TREM2 gene have been associated with amyloidogenesis in neurodegenerative diseases including AD. In this report, we provide evidence that aluminum-sulfate, when incubated with microglial cells, induces the up-regulation of an NF-кB-sensitive micro RNA-34a (miRNA-34a; chr1p36) that is known to target the TREM2 mRNA 3'-untranslated region (3'-UTR), significantly down-regulating TREM2 expression. The aluminum-induced up-regulation of miRNA-34a and down-regulation of TREM2 expression were effectively quenched using the natural phenolic compound and NF-kB inhibitor CAPE [2-phenylethyl-(2E)-3-(3,4-dihydroxyphenyl) acrylate; caffeic-acid phenethyl ester]. These results suggest, for the first time, that an epigenetic mechanism involving an aluminum-triggered, NF-kB-sensitive, miRNA-34a-mediated down-regulation of TREM2 expression may impair phagocytic responses that ultimately contribute to Aβ42 peptide accumulation, aggregation, amyloidogenesis and inflammatory degeneration in the brain.

Keywords: Aluminum sulfate; Alzheimer's disease; Genotoxicity; Inflammation; Microglial cells; Phagocytosis; TREM2.

Figure 1

(A) CB-84 (ATCC CRL-2467) murine microglial cells, 32% confluent, phase contrast microscopy; bar = 20 μm; (B) fluorescent-based miRNA array analysis results for 3 magnesium sulfate-treated and 3 aluminum-sulfate-treated experiments using CB-84 murine microglial cells [as described in (A)]; compared to 2 uM ambient magnesium, 2 uM ambient aluminum induces a small family of micro RNAs (miRNAs) in murine microglial cells; these include miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155 but not miRNA-183; up-regulation of these induced miRNAs has been previously shown to be NF-kB-sensitive [–23]; microRNAs (miRNAs) are a recently discovered class of single stranded non-coding ribonucleotide regulators which, through base-pair complementarity, bind to the 3′ un-translated (3′-UTR) region of highly selective target mRNAs, and direct the post-transcriptional repression of that mRNA’s encoded genetic information [25,26] (Fig. 1B); the aluminum-mediated up-regulation of miRNA-9, miRNA-146a, miRNA-125b and miRNA-155 and their pathogenic consequences have already been reported [,–25,28,33,37]; miRNA-183 is a control miRNA whose levels do not change in the presence of either magnesium or aluminum in microglial or other brain cell types [11,18,22]; (C) Quantitation of fluorescent signals in (B); miRNA-34a (highlighted in a yellow rectangle) is up-regulated 2.6-fold in aluminum-sulfate-treated microglial cells (compared to magnesium sulfate-treated controls); note that treatments longer than 8 hrs with <2 uM ambient aluminum gave quantitatively similar results [23]; addition of caffeic acid phenethyl ester (CAPE), a potent honeybee-resin (propolis)-derived NF-kB inhibitor quenched this induction, indicating the NF-kB-sensitivity of miRNA-34a expression; (D) within the same microglial cells TREM2 protein abundance is shown in comparison to an unchanging control β-actin protein abundance in the same sample; (representative protein bands, upper panel; quantified in bar format, lower panel); TREM2 is significantly down-regulated to 0.24-fold of control values; again, addition of CAPE inhibitor quenched this induction, indicating that TREM2 up-regulation is NF-kB-sensitive. An aluminum-induced reduction in TREM2 expression may therefore impair phagocytosis of Aβ42 peptides with amyloidogenic effects (see text); a dashed horizontal line at 1.0 indicates in (C) control miRNA-34a levels or (D) control TREM2 protein levels for ease of comparison; figures were generated using Adobe Photoshop v9.0 (Adobe, San Jose CA, USA); statistical procedures were analyzed using a two-way factorial analysis of variance (p, ANOVA) and the SAS language (Statistical Analysis Institute, Cary NC, USA) [11,21]. Only p-values less than 0.05 (ANOVA) were considered as statistically significant; N=5, significance over controls *p<0.05 (ANOVA).