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. 2012;7(5):e35921.
doi: 10.1371/journal.pone.0035921. Epub 2012 May 15.

Reduced mature microRNA levels in association with dicer loss in human temporal lobe epilepsy with hippocampal sclerosis

Ross C McKiernan  1 , Eva M Jimenez-Mateos, Isabella Bray, Tobias Engel, Gary P Brennan, Takanori Sano, Zuzanna Michalak, Catherine Moran, Norman Delanty, Michael Farrell, Donncha O'Brien, Robert Meller, Roger P Simon, Raymond L Stallings, David C Henshall
Affiliations

Reduced mature microRNA levels in association with dicer loss in human temporal lobe epilepsy with hippocampal sclerosis

Ross C McKiernan et al. PLoS One. 2012.

Abstract

Hippocampal sclerosis (HS) is a common pathological finding in patients with temporal lobe epilepsy (TLE) and is associated with altered expression of genes controlling neuronal excitability, glial function, neuroinflammation and cell death. MicroRNAs (miRNAs), a class of small non-coding RNAs, function as post-transcriptional regulators of gene expression and are critical for normal brain development and function. Production of mature miRNAs requires Dicer, an RNAase III, loss of which has been shown to cause neuronal and glial dysfunction, seizures, and neurodegeneration. Here we investigated miRNA biogenesis in hippocampal and neocortical resection specimens from pharmacoresistant TLE patients and autopsy controls. Western blot analysis revealed protein levels of Dicer were significantly lower in certain TLE patients with HS. Dicer levels were also reduced in the hippocampus of mice subject to experimentally-induced epilepsy. To determine if Dicer loss was associated with altered miRNA processing, we profiled levels of 380 mature miRNAs in control and TLE-HS samples. Expression of nearly 200 miRNAs was detected in control human hippocampus. In TLE-HS samples there was a large-scale reduction of miRNA expression, with 51% expressed at lower levels and a further 24% not detectable. Primary transcript (pri-miRNAs) expression levels for several tested miRNAs were not different between control and TLE-HS samples. These findings suggest loss of Dicer and failure of mature miRNA expression may be a feature of the pathophysiology of HS in patients with TLE.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reduced Dicer levels in TLE patients with hippocampal sclerosis.
(A) Representative histology for autopsy controls and patient samples. (a) Photomicrograph showing field view of a cresyl violet-stained section from an autopsy control hippocampus (C2). (b) Photomicrograph showing a field view of the hippocampus from a TLE-HS patient (E2). Boxes in b indicate regions of the section from which higher-power images and other stains in c–e are presented. (c) Evident pyramidal neuron loss in the CA1 sector (20× lens). (d) Granule cell layer dispersion in E2. (e) GFAP immunohistochemistry showing astrogliosis within the granule cell layer. (f, g) Cresyl violet and GFAP-stained sections from patient E3 showing granule cell layer dispersion and astrogliosis (20× lens). Scale bar in a, b, 1 mm; c–g, 100 µm. (B) Representative Western blots showing protein levels of neuron (NeuN) and astrocyte (GFAP) markers in hippocampus from controls (C1–4) and TLE-HS patients (E1–4). (C) Semi-quantification of protein levels (n = 4 per group). Levels of the uppermost band (∼66 kD) and a lower band (48 kD) were lower in TLE-HS samples, whereas GFAP (uppermost band analyzed) was higher. (D, E) Representative Western blots and graphs showing protein levels and semi-quantification for Dicer, Drosha and AGO2. *p<0.05; **p<0.01 compared to controls. Protein levels corrected to α-Tubulin (α-Tub).
Figure 2
Figure 2. Reduced mature miRNA expression in human TLE-HS.
(A) Heat-map depiction of raw miRNA expression data for 188 of the expressed miRNAs from controls (C1–C4) and TLE-HS samples (TLE1, TLE2 and TLE4). Note the reduction in miRNA levels (green bars) in TLE-HS samples. Scale bar to left indicates fold differences. (B) Pie chart summarizing the findings from profiling miRNA levels in human TLE-HS samples. Over half the expressed miRNAs were found to be lower than in controls. (C) Graph showing the expression of the significantly down-regulated miRNAs in TLE-HS samples. Individual Ct values from each TLE sample were normalized to the average of the Ct values from the control group. The graph represents the average of the 2−ΔΔCt of each TLE sample. *p<0.05; **p<0.01; ***p<0.001 compared to control (from n = 4 controls versus n = 3 TLE-HS samples). Note, Hsa is nomenclature denoting human miRNA sequence. (D) Graphs (n = 3–4 per group) showing expression (2−ΔΔCt, normalized to Gapdh) for a selection of pri-miRNAs in the same control and TLE-HS samples.
Figure 3
Figure 3. miRNA biogenesis components in neocortex and in experimental epilepsy.
(A) Representative Western blots showing expression of Dicer, Drosha and AGO2 in neocortex samples from TLE patients (TLE 9–11) and controls (C3–C5). (B) Graphs (n = 3 per group) showing semi-quantification of levels of each protein. **p<0.05 compared to controls. Protein levels corrected to α-Tubulin (α-Tub). (C) Representative immunohistochemistry illustrating the hippocampal damage in epileptic mice. (a) Field view (4× lens) showing triple-stained section from an epileptic mouse 21 days after status epilepticus. Box highlights main lesion area in CA3. b,c are 20× lens field views of the lesioned CA3 area showing reduced NeuN (red) and increased GFAP (green) staining. (D) Representative views of the CA3 from a control mouse at 21 days which did not undergo status epilepticus. a–c show NeuN (red), GFAP (green) and a merge of these with DAPI overlay. Note the intact neuron population and minimal GFAP staining. Scale bar in Ca, 500 µm; Cb, and Da, 100 µm. (E, F) Representative Western blots (n = 1 per lane) showing protein levels of NeuN, GFAP, Dicer, Drosha and Ago2 in CA3 samples from control and epileptic (21 day) mice (TLE-HS). (G) Graph quantifying reduction in hippocampal CA3 Dicer levels in epileptic animals (n = 7–8 per group; *p<0.05 compared to control). Protein levels corrected to α-Tub. (H) Representative Western blots (n = 1 per lane) show non-damaging seizures do not cause a reduction of Dicer levels. α-Tubulin is included as a guide to protein loading.
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