miRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.036
ISSN1525-2191
AutoresEva M. Jiménez‐Mateos, Isabella Bray, Amaya Sanz‐Rodriguez, Tobías Engel, Ross C. McKiernan, Genshin Mouri, Katsuhiro Tanaka, Takanori Sano, Julie A. Saugstad, Roger P. Simon, Raymond L. Stallings, David C. Henshall,
Tópico(s)Circular RNAs in diseases
ResumoWhen an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death. When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death. The brain possesses endogenous molecular mechanisms by which it can protect itself from harm, although forewarning is required to bring protection optimally to bear. Thus, mild noxious stressors such as brief ischemia or brief seizures evoke protective adaptations in the brain, which render it powerfully refractory against a subsequent and otherwise damaging insult.1Gidday J.M. Cerebral preconditioning and ischaemic tolerance.Nat Rev Neurosci. 2006; 7: 437-448Crossref PubMed Scopus (640) Google Scholar, 2Dirnagl U. Becker K. Meisel A. 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Matsushima S. Taki W. Prehn J.H. Simon R.P. Henshall D.C. Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.Neuroscience. 2007; 150: 467-477Crossref PubMed Scopus (43) Google Scholar We then used a small-molecule-inhibitor approach to model the changes brought on by tolerance and to test whether miRNAs contribute in vivo to seizure-induced neuronal death. All animal experiments were performed in accordance with European Communities Council Directive 86/609/EEC and were reviewed and approved by the Research Ethics Committee of the Royal College of Surgeons in Ireland, under license from the Department of Health, Dublin, Ireland. Adult male C57BL/6 mice (20 to 22 g) from Harlan (Oxon, Bicester, UK) were used. Control mice received a single intraperitoneal injection of 0.2 mL saline on day 1 (sham preconditioning), followed by intra-amygdalar vehicle on day 2 (0.2 μL PBS). Injury group mice also received intraperitoneal injection of 0.2 mL saline on day 1, and then underwent status epilepticus induced by intra-amygdalar KA (Sigma-Aldrich, Dublin, Ireland; St. Louis, MO) on day 2. Tolerance mice underwent seizure preconditioning on day 1 via intraperitoneal injection of 0.2 mL KA (15 mg/kg), and then underwent status epilepticus on day 2 induced by intra-amygdalar KA. For intra-amygdalar injections, mice were anesthetized with isoflurane (5% induction, 1% to 2% maintenance) and placed in a mouse-adapted stereotaxic frame. Body temperature was maintained within the normal physiological range with a rectal thermometer and feedback-controlled heat pad (Harvard Apparatus, Kent, UK; Holliston, MA). After a midline scalp incision, the bregma was located and three partial craniectomies were performed for placement of skull-mounted recording screws (Bilaney Consultants, Sevenoaks, Kent, UK). A complete craniectomy was drilled for placement of a guide cannula for intra-amygdalar injections (coordinates from bregma: AP = −0.94 mm, L = −2.85 mm), based on a stereotaxic atlas.33Paxinos G. Franklin K.B.J. The mouse brain in stereotaxic coordinates. ed 2. Elsevier, San Diego2001Google Scholar The cannula and electrode assembly was fixed in place with dental cement and the animal was placed in a recording chamber. The electroencephalogram (EEG) was recorded using a Grass Comet XL laboratory-based EEG system (Grass Technologies, Slough, UK; West Warwick, RI). After baseline EEG recordings, the animal was lightly restrained while an injection cannula was lowered 3.75 mm below the brain surface for injection of KA (1 μg) or vehicle in a volume of 0.2 μL into the basolateral amygdala nucleus. After 40 minutes, all mice received lorazepam (Ativan; 6 mg/kg, i.p.). Animals were recorded for up to 1 hour thereafter, before being disconnected and placed in a warmed recovery chamber. For intracerebroventricular injections, additional mice were affixed with a cannula ipsilateral to the site of KA injection, as described previously.34Murphy B.M. Engel T. Paucard A. Hatazaki S. Mouri G. Tanaka K. Tuffy L.P. Jimenez-Mateos E.M. Woods I. Dunleavy M. Bonner H.P. Meller R. Simon R.P. Strasser A. Prehn J.H. Henshall D.C. Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus.Cell Death Differ. 2010; 17: 459-468Crossref PubMed Scopus (36) Google Scholar Coordinates from the bregma were AP = −0.3 mm, L = −1.0 mm, V = −2.0 mm. Mice received 2 μL infusion of either scrambled or miR-132 antagomir (locked nucleic acid LNA- and 3′-cholesterol-modified oligonucleotides; Exiqon, Vedbaek, Denmark) in artificial cerebrospinal fluid (Harvard Apparatus). Twenty-four hours later, mice were either euthanized or were subjected to status epilepticus, as described above. Mice were euthanized at 24 hours after intra-amygdalar injections. Animals were given a pentobarbital overdose and were perfused with ice-cold saline to remove intravascular blood components. Brains for molecular and biochemical work were dissected on dry ice under a microscope and the hippocampus was subdivided to obtain the separate CA3-enriched portion, as described previously.8Jimenez-Mateos E.M. Hatazaki S. Johnson M.B. Bellver-Estelles C. Mouri G. Bonner C. Prehn J.H. Meller R. Simon R.P. Henshall D.C. Hippocampal transcriptome after status epilepticus in mice rendered seizure damage-tolerant by epileptic preconditioning features suppressed calcium and neuronal excitability pathways.Neurobiol Dis. 2008; 32: 442-453Crossref PubMed Scopus (65) Google Scholar For histology, mice were first perfused with ice-cold saline and then either perfusion-fixed with paraformaldehyde (4%) or brains were fresh-frozen in 2-methylbutane at −30°C. EEGs were analyzed using TWin software v3.8 (Grass Technologies), and the duration of high-amplitude, high-frequency discharges (also termed type IV seizures) was calculated between the time of KA injection and the time of lorazepam administration.35Engel T. Hatazaki S. Tanaka K. Prehn J.H. Henshall D.C. Deletion of Puma protects hippocampal neurons in a model of severe status epilepticus.Neuroscience. 2010; 168: 443-450Crossref PubMed Scopus (21) Google Scholar Additional frequency and amplitude analysis of EEG was performed using LabChart Pro version 7 software (ADInstruments, Oxford, UK). Total mRNA was extracted using an miRNeasy kit (Qiagen, West Sussex, UK; Valencia, CA) according to the manufacturer's instructions to obtain an enrichment of small RNAs. For each condition, ipsilateral CA3 subfields from three separate mice were pooled together for analysis, and this was repeated using independent samples. Quality and quantity of mRNA was measured using a NanoDrop spectrophotometer (Thermo Scientific, Loughborough, UK; Wilmington, DE), and RNA dilutions were made up in nuclease-free water. Reverse transcription of 250 ng of miRNA from the CA3 subfields from each condition was performed using stem-loop multiplex primer pools (Applied Biosystems, Paisley, UK; Foster City, CA), allowing reverse transcription of 48 different miRNAs in each of eight RT pools. The miRNA quantitative PCR (qPCR) screen was performed on the 7900HT fast real-time system using TaqMan low-density arrays (TaqMan TLDA MicroRNA assays version 1.0 containing 380 human microRNAs assays; Applied Biosystems). Of the profiled human miRNAs, 197 were 100% homologous to mouse. miRNAs were considered differentially expressed at a threshold of ±1.5-fold.36Liu D.Z. Tian Y. Ander B.P. Xu H. Stamova B.S. Zhan X. Turner R.J. Jickling G. Sharp F.R. Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures.J Cereb Blood Flow Metab. 2010; 30: 92-101Crossref PubMed Scopus (433) Google Scholar Reverse transcription for individual qPCRs was performed using 250 ng of total RNA and a high-capacity reverse transcription kit (Applied Biosystems); RT-specific primers for mouse miRNAs miR-27a, miR-92a, miR-101, miR-127, miR-132, miR-145, miR-200a, and miR-326 (Applied Biosystems) were used for all miRNA reverse transcription. Individual qPCRs were performed on the 7900HT fast real-time system (Applied Biosystems) using miR-27a, miR-92a, miR-101, miR-127, miR-132, miR-145, miR-200a, and miR326-specific TaqMan microRNA assays (Applied Biosystems). RNU19 was used for normalization miRNA expression studies, as described previously.37Shibata M. Nakao H. Kiyonari H. Abe T. Aizawa S. MicroRNA-9 regulates neurogenesis in mouse telencephalon by targeting multiple transcription factors.J Neurosci. 2011; 31: 3407-3422Crossref PubMed Scopus (265) Google Scholar A relative fold change in expression of the target gene transcript was determined using the comparative cycle threshold method (2−ΔΔCT). Western blot analysis was performed as described previously.38Engel T. Murphy B.M. Hatazaki S. Jimenez-Mateos E.M. Concannon C.G. Woods I. Prehn J.H. Henshall D.C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.FASEB J. 2010; 24: 853-861Crossref PubMed Scopus (55) Google Scholar Hippocampal CA3 subfields were homogenized in a lysis buffer, boiled in gel-loading buffer, separated on SDS-PAGE gels, and transferred onto nitrocellulose membranes. The following primary antibodies were used: Drosha (1:400; Cell Signaling Technology, Danvers, MA), Dicer-1 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA), Argonaute-2 (Ago-2) (1:400; Cell Signaling Technology), and Tubulin (1:2000, Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and bands were visualized using Pierce SuperSignal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL). Images were captured using a Fuji-Film LAS-300 (Fuji, Sheffield, UK), and densitometry was performed using AlphaEaseFC4.0 gel-scanning integrated optical density software (Alpha Innotech; ProteinSimple, San Leandro, CA). Pools of three individual mouse CA3 subfields from each condition were homogenized in 0.7 mL of ice-cold immunoprecipitation buffer (300 mmol/L NaCl, 5 mmol/L MgCl2, 0.1% NP-40, 50 mmol/L Tris-HCl pH 7.5). The homogenate was centrifuged at 16,000 × g for 15 minutes at 4°C and the supernatant was considered as the total cell lysate. Five micrograms of anti-Ago-2 was added to each 400 μg of supernatant in a final volume of 1 mL. The solution was vortex-mixed and incubated overnight at 4°C. After addition of 20 μL of 50% protein-A/G-agarose beads (Santa Cruz Biotechnology), the solution was mixed and incubated for 1 hour at 4°C. The beads were centrifuged at 16,000 × g for 15 minutes at 4°C, and the supernatant was removed. The pellet was washed twice with immunoprecipitation buffer, and miRNA was extracted using an miRNeasy kit (Qiagen, West Sussex, UK). Stem-loop reverse transcription and real-time qPCR (Applied Biosystems) was performed as described above to semiquantify the expression of the miRNA. To characterize seizure-damage in the model, brains from control, injury, and tolerance mice were sectioned at 12 μm on a cryostat (−20°C) at the level of either rostral (AP = −1.58 mm) or medial (AP = −1.82 mm) hippocampus.33Paxinos G. Franklin K.B.J. The mouse brain in stereotaxic coordinates. ed 2. Elsevier, San Diego2001Google Scholar Fluoro-Jade B (FJB) staining was performed as described previously.8Jimenez-Mateos E.M. Hatazaki S. Johnson M.B. Bellver-Estelles C. Mouri G. Bonner C. Prehn J.H. Meller R. Simon R.P. Henshall D.C. Hippocampal transcriptome after status epilepticus in mice rendered seizure damage-tolerant by epileptic preconditioning features suppressed calcium and neuronal excitability pathways.Neurobiol Dis. 2008; 32: 442-453Crossref PubMed Scopus (65) Google Scholar, 39Jimenez-Mateos E.M. Mouri G. Conroy R.M. Henshall D.C. Epileptic tolerance is associated with enduring neuroprotection and uncoupling of the relationship between CA3 damage, neuropeptide Y rearrangement and spontaneous seizures following intra-amygdalar kainic acid-induced status epilepticus in mice.Neuroscience. 2010; 171: 556-565Crossref PubMed Scopus (18) Google Scholar Briefly, tissue sections were allowed to air-dry and then were postfixed in formalin (10%), immersed in 0.006% potassium permanganate solution, rinsed again, and then transferred to FJB solution (0.001% in 0.1% acetic acid) (Millipore-Chemicon Europe, Chandlers Ford, UK; Billerica, MA). Sections were then rinsed again, dried, cleared, and mounted in DPX mounting medium (Sigma-Aldrich). Analysis of DNA damage was performed on fresh-frozen sections using a fluorescein-based TUNEL technique, according to the manufacturer's instructions (Promega, Madison, WI) and as described previously.38Engel T. Murphy B.M. Hatazaki S. Jimenez-Mateos E.M. Concannon C.G. Woods I. Prehn J.H. Henshall D.C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.FASEB J. 2010; 24: 853-861Crossref PubMed Scopus (55) Google Scholar For assessment of neuron loss, sections were fixed, blocked in 5% goat serum, and incubated with antibodies against neuronal nuclear protein (1:500 NeuN; Millipore Ireland, Tullagreen, Cork, Ireland), which was detected using goat anti-mouse Alexa Fluor 568 (Bio-Sciences, Dun Laoghaire, Ireland). Sections were examined using a Nikon 2000s epifluorescence microscope (Micron Optical, Enniscorthy, Ireland) under excitation/emission (Ex/Em) wavelengths of 472/520 nm (green) and 540 to 580/600 to 660 nm (red). Pseudocolor transforms from monochromatic images acquired using an Orca-285 digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) were generated using Adobe Photoshop version 6.0 software. Cell counts were performed for the entire CA3 subfield, beginning at the border with CA2 through to the end of CA3c/CA4 within the hilus of the dentate gyrus. Counts of FJB-, TUNEL- and NeuN-stained cells were the average of two adjacent sections assessed by an observer blinded to experimental group and condition. For in situ hybridization, mice (n = 3) were perfused with ice-cold PBS followed by paraformaldehyde (4%). Sections (12 μm thick) were mounted on SuperFrost-Plus slides (VWR International, Radnor, PA) and air dried. Using RNase free solutions, slides were washed with PBS and radioimmunoprecipitation assay buffer [150 mL NaCl, 1% (octylphenoxy)polyethoxyethanol (IGEPAL)], 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, 50 mmol/L Tris pH 8.0) for 5 minutes, then treated with 4% paraformaldehyde for 10 minutes. Sections were washed again and treated with 0.25% acetic anhydride/0.1 mol/L triethanolamine, then rinsed with 0.1% Tween-20/PBS for 5 minutes, treated with 5 μg/mL proteinase K for 4 minutes, and washed with PBS. Next, slides were rinsed in prehybridization buffer (1× saline solution, 50% formamide, and 1× Denhardt's solution) for 1 hour at 56°C (melting temperature Tm = −20°C, as specified by Exiqon). The probe to detect mi-132 was 5′-digoxigenin-labeled, 2′-O,4′-C-methylene bicyclonucleoside monomer-containing oligonucleotide (LNA-modified). The sequence was the reverse complement to the mature miRNA. Probes were incubated 1:200 in hybridization buffer (1× saline solution, 50% formamide, and 1× Denhardt's plus 10% dextran sulfate) overnight at 56°C in a humidified chamber. On the next day, sections were washed in FAM buffer (2× SSC, 50% formamide, and 0.1% Tween 20) for 1 hour at 60°C. Then sections were rinsed in B1 buffer (150 mmol/L NaCl, 100 mmol/L maleic acid, and 0.4% IGEPAL pH, 7.5) for 1 hour at room temperature and in B2 buffer (2% blocking reagent and 10% goat serum in B1 buffer) for 30 minutes. Anti-DIG-PA antibody (1:1000; Roche Applied Science, Indianapolis, IN) was incubated in B2 buffer overnight at 4°C. On the next day, sections were washed in B1 buffer and incubated in B3 buffer (100 mmol/L NaCl, 50 mmol/L MgCl2, 0.025% Tween 20 and 100 mmol/L Trizma pH9.5) for 30 minutes. Then, 200 μL of color substrate solution [nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (BNT/BCIP stock solution; Roche Applied Science)] diluted 1:50 in B3 buffer was added to each slide until the sign
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