Upregulation of micro RNA-146a (miRNA-146a), a marker for inflammatory neurodegeneration, in sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker (GSS) syndrome

Abstract

A mouse- and human-brain-abundant, nuclear factor (NF)-кB-regulated, micro RNA-146a (miRNA-146a) is an important modulator of the innate immune response and inflammatory signaling in specific immunological and brain cell types. Levels of miRNA-146a are induced in human brain cells challenged with at least five different species of single- or double-stranded DNA or RNA neurotrophic viruses, suggesting a broad role for miRNA-146a in the brain's innate immune response and antiviral immunity. Upregulated miRNA-146a is also observed in pro-inflammatory cytokine-, Aβ42 peptide- and neurotoxic metal-induced, oxidatively stressed human neuronal-glial primary cell cocultures, in murine scrapie and in Alzheimer's disease (AD) brain. In AD, miRNA-146a levels are found to progressively increase with disease severity and co-localize to brain regions enriched in inflammatory neuropathology. This study provides evidence of upregulation of miRNA-146a in extremely rare (incidence 1-10 per 100 million) human prion-based neurodegenerative disorders, including sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). The findings suggest that an upregulated miRNA-146a may be integral to innate immune or inflammatory brain cell responses in prion-mediated infections and to progressive and irreversible neurodegeneration of both the murine and human brain.

FIGURE 1

(A) Homo sapiens micro RNA-146a (hsa-miRNA-146a) is a 22-nucleotide small RNA (highlighted in yellow) abundant in mouse and human immune cells and the limbic system, with known mRNA 3′ UTR targets that include the interleukin-1 receptor associated kinase 1 (IRAK-1; highlighted in red); uppercase ribonucleotides involved in fully (|) or partially (:) complementary hydrogen bonding (Cui et al. 2010; Taganov et al. 2006); chromosome location of the human IRAK-1 gene (chrXq28) and Genbank accession (NM_01569.3) indicated; E_A = predicted energy of association for miRNA-mRNA interaction (Sethi and Lukiw 2009; miRBASE, Cambridge, UK); the mouse and human miRNA-146a are identical in sequence (Li et al. 2010a; 2010b; Sethi and Lukiw 2009). (B) Structural features of the miRNA-146a encoding DNA locus at chromosome 5q33.3 and details of the NF-κB-sensitive miRNA146a gene showing three upstream (5′) regulatory NF-КB binding sites; miRNA-146a transcription is highly NF-КB sensitive (Cui et al. 2010; Lukiw et al. 2008; Taganov et al. 2006). (C) The pre-miRNA-146a transcribed from the miRNA146a locus (chr 5q33.3) has strong potential to form a highly stable 35-base-pair stem, 60-nucleotide loop RNA structure (stem-loop E_A= −49.5 kcal/mol); other predicted secondary structures of alternate stem-loop configurations are possible and the 5′ and 3′ ends of pre-miRNA-146a may be significantly extended. In several preferential E_A models, such as the one depicted in (C), the stem-loop structure containing the miRNA-146a sequence is consistently located in the very distal end of the predicted loop (highlighted in yellow; delineated by arrows), after which Dicer (RNase III) processing of this pre-miRNA-146a (Yokota 2009) yields the mature miRNA-146a highlighted in yellow in (A) (color figure available online).

FIGURE 2

Abundance of hsa-miRNA-125b and/or hsa-miRNA-146a in comparison to an internal control hsa-5S RNA marker in murine scrapie 139a, sporadic Creutzfeldt-Jakob disease (sCJD), and Gerstmann-Straussler-Scheinker syndrome (GSS) compared to age-matched controls (control) in the same brain region as analyzed by (A) fluorescent miRNA array cluster analysis (LC Sciences, Houston TX) and (B) concentrated Northern dot-blot analysis of 5S RNA and miRNA-146a using high-specific-activity radiolabeled probes (Cui et al. 2005; Sethi and Lukiw 2009); (C) quantified results from Northern dot-blot analysis; dashed horizontal line indicates control 139A levels relative to 5S RNA for ease of comparison. In (A) panel 1 is the age-matched control for murine 139a (n= 2; lanes 4 and 5); lanes 2 and 3 are the mean age-matched control for the individual cases for sCJD (n = 3; lanes 6–8) and GSS (n= 2, lanes 9 and 10), respectively; by convention, green colors indicate no change and red colors indicate upregulation (LC Sciences; Lukiw et al. 2008). Squared black lines to the left of panel (A) indicate that a mean signal intensity comparison between hsa-5S–RNAand hsa-miRNA-125b yielded a significance of p > .07 (ANOVA), and between 5S–RNA and hsa-miRNA-146a yielded a p< .01 (ANOVA). miRNA-125b was found to be significantly upregulated in one of two GSS cases and two of three CJD cases; miRNA-146a was found to be consistently elevated in all sCJD or GSS cases using miRNA array analysis; and this was further confirmed using quantitative Northern dot blot analysis (B and C). The 22 nucleotide hsa-5S–RNA probe was derived from the 5′ end of the 107 nucleotide human 5S ribosomal RNA (5S RNA) and in panel (B) was loaded at 1/10 the concentration of miRNA-146a (Sethi and Lukiw 2009) (color figure available online).