microRNA (miRNA) speciation in Alzheimer's disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Abstract

Human cerebrospinal fluid (CSF), produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease. In these experiments we analyzed amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in CSF and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer's disease (AD) and age-matched control neocortex. There was a trend for decreased abundance of Aβ42 in the CSF and ECF in AD but it did not reach statistical significance (mean age ~72 yr; N=12; p~0.06, ANOVA). The most abundant nucleic acids in AD CSF and ECF were miRNAs, and their speciation and inducibility were studied further. Fluorescent miRNA-array-based analysis indicated significant increases in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 in AD CSF and ECF (N=12; p<0.01, ANOVA). Primary human neuronal-glial (HNG) cell co-cultures stressed with AD-derived ECF also displayed an up-regulation of these miRNAs, an effect that was quenched using the anti-NF-кB agents caffeic acid phenethyl ester (CAPE) or 1-fluoro-2-[2-(4-methoxy-phenyl)-ethenyl]-benzene (CAY10512). Increases in miRNAs were confirmed independently using a highly sensitive LED-Northern dot-blot assay. Several of these NF-кB-sensitive miRNAs are known to be up-regulated in AD brain, and associate with the progressive spreading of inflammatory neurodegeneration. The results indicate that miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 are CSF- and ECF-abundant, NF-кB-sensitive pro-inflammatory miRNAs, and their enrichment in circulating CSF and ECF suggest that they may be involved in the modulation or proliferation of miRNA-triggered pathogenic signaling throughout the brain and central nervous system (CNS).

Keywords: Alzheimer’s disease (AD); CAPE (caffeic acid phenethyl ester); CAY10512 (1-fluoro-2-[2-(4-methoxyphenyl)-ethenyl]-benzene); NF-кB; inflammatory signaling; micro RNA.

Figure 1

miRNA array analysis of control and AD ECF and CSF; A: human neocortical extracellular fluid (ECF; derived from high speed AD and age-matched tissue supernatants) or cerebrospinal fluid (CSF) were analyzed for miRNA speciation and mean abundance using fluorescent reporter miRNA arrays [13,19-23]. miRNA array data for 6 of the most abundant miRNAs found in AD ECF or CSF (N=3 representative samples shown) compared to age-matched controls (N=3 samples shown) are shown in [A] and quantified in [B]; these extracellular fluids exhibit elevations in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 and other AD-enriched small non-coding RNAs (sncRNAs; miRNA-34a, miRNA-128) compared to age-matched controls or unchanging miRNAs such as miRNA-183 [13,20]. Postmortem intervals (PMIs) for age-matched control or AD human brain tissue ECF were all less than 3 hr; all tissues were from the medial temporal lobe neocortex; the study group (controls N=6; AD N=6) of tissues exhibited no significant differences in age (mean plus one standard deviation = 72.4±6.5 vs 72.5±7.5 yr, p<0.85), PMI (mean 2.1±2.1 vs 2.2±1.4 hr, p<0.95), RNA A_260/280 indices (2.1±0.5 vs 2.05±0.4, p<0.95) or RNA 28S/18S (1.5 vs 1.45, p<0.92) age-matched control and AD, respectively. RNA integrity numbers (RIN) were ≥ 8.8; no significant differences in total RNA yield between the control and AD groups were noted; in [B] a dashed horizontal line at 1.0 indicates a control miRNA-183 signal in the ECF for ease of comparison. miRNAs were analyzed by LC Sciences (Houston, TX) using miRNA array panels containing 1898 individual human miRNA targets; N=6, *p<0.01 (ANOVA) [16-19].

Figure 2

Analysis of miRNA in ECF-stressed HNG cells; A: human neuronal-glial (HNG) primary cells 2 weeks in co-culture; 20x; cells are triple stained using antibodies to glial fibrillary acidic protein (GFAP), a glial-specific marker (green fluorescence; λ_max=556 nm), with βTUBIII, a neuron-specific marker (red; λ_max=702 nm) and Hoescht 33258 to highlight cellular nuclei (blue; λ_max=461 nm) [3,11-13,19-23]; human primary neurons do not culture well by themselves so are co-cultured with primary glial cells [21,22]; B: HNG cells were stressed with AD-derived ECF (containing ~30 ug total protein; see text) for 5 time-points (0, 24, 48, 72 and 96 hrs); no additional increases were observed after 72 hrs of ECF treatment; LED-Northern dot blots of miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 in control (0 hr) and AD ECF-stressed (72 hr) HNG cells using an unchanging miRNA-183 as an internal control marker; C: LED-Northern-based graphed results of miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 and a control miRNA-183 in ECF-stressed HNG cells indicating relative levels at 0 (control), 24 and 72 hrs; dashed horizontal line at 1.0 indicates miRNA-183 control levels for ease of comparison; N=6; *p<0.05; **p<0.01 (ANOVA).

Figure 3

Down-regulation of miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 by the NF-кB inhibitors CAPE and CAY10512; the presence of caffeic acid phenethyl ester (CAPE; Sigma-Aldrich, St. Louis MO), an active component of an organic resinous mixture that honey bees collect from botanical sources, or the polyphenolic transstilbene resveratrol analog CAY10512 down-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 induction to ~0.1- to ~0.25-fold of controls strongly suggesting that these inducible miRNAs are derived from NF-кB-regulated genes; [13, 21, 29; see text; unpublished); N=5; *p<0.01 (ANOVA).