Ischemic preconditioning regulates expression of microRNAs and a predicted target, MeCP2, in mouse cortex

Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon 97232, USA.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism (Impact Factor: 5.41). 12/2009; 30(4):744-56. DOI: 10.1038/jcbfm.2009.253
Source: PubMed
ABSTRACT
Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective response that protects the brain during a subsequent severe ischemic injury, a phenomenon known as 'tolerance'. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming of the brain's response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA) translation. We examined miRNA expression in mouse cortex in response to preconditioning, ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA expression is consistently altered within each group, but that preconditioning was the foremost regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in preconditioned cortex. Downregulation of miR-132 is consistent with our finding that preconditioning ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors of ischemic preconditioning-induced tolerance.

Full-text

Available from: Giuseppe Pignataro, Feb 12, 2014
Ischemic preconditioning regulates expression of
microRNAs and a predicted target, MeCP2, in
mouse cortex
Theresa A Lusardi, Carol D Farr, Craig L Faulkner, Giuseppe Pignataro, Tao Yang,
Jingquan Lan, Roger P Simon and Julie A Saugstad
Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon, USA
Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective
response that protects the brain during a subsequent severe ischemic injury, a phenomenon known
as ‘tolerance’. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming
of the brain’s response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate
posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA)
translation. We examined miRNA expression in mouse cortex in response to preconditioning,
ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA
expression is consi stently altered within each group, but that preconditioning was the foremost
regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated
miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG
binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to
preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in
preconditioned cortex. Downregulation of miR-132 is consistent with our finding that precondition-
ing ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These
studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted
targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors
of ischemic preconditioning-induced tolerance.
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744–756; doi:10.1038/jcbfm.2009.253; published online
16 December 2009
Keywords:
ischemic preconditioning; microRNA; microarray; MeCP2; tolerance
Introduction
Ischemic brain injuries are among the most common
and important causes of disability and death world-
wide. However, a sublethal duration of ischemia,
ischemic preconditioning, triggers endogenous re-
sponses that protect the brain against a subsequent
severe ischemic insult, a phe nomenon known as
‘tolerance’ (Dirnagl et al, 2009). The mechanisms of
preconditioning-induced tolerance are not well
known, but are characterized by three key features.
Ischemic tolerance requires de novo protein synth-
esis (Barone et al, 1998), is correlated with repressed
gene expression (Bowen et al, 2006; Koerner et al,
2007; Stenzel-Poore et al, 2003), and is transient
(Chen et al, 1996; Perez-Pinzon et al, 1997).
MicroRNAs (miRNAs) regulate posttranscriptional
gene expression in plants, animals, and viruses
(Ambros, 2004; Bartel, 2004; Chen and Meister,
2005) and are integral components of RNA-induced
silencing complexes, which repress translation by
directly interacting with messenger RNAs (mRNAs).
In animals, miRNAs regulate mRNA translation
through an imperfect pairing with nucleotide
sequences within the 3
0
untranslated region (3
0
UTR)
of targets, and repressed translation is enhanced for
those mRNAs targeted by multiple miRNAs (Doench
and Sharp, 2004). MiRNAs repressing translation are
sequestered and localized in processing bodies (Pillai
et al, 2005; Sheth and Parker, 2003); release of
miRNA-targeted mRNAs sequestered in processing
bodies can occur without affecting mRNA stability
(Liu et al, 2005; Pillai et al, 2005; Sen and Blau,
2005). Cellular stress can cause the release of mRNA
from processing bodies, leading to recruitment of
Received 10 August 2009; revised 24 September 2009; accepted 10
November 2009; published online 16 December 2009
Correspondence: JA Saugstad, Senior Scientist, Robert S. Dow
Neurobiology Laboratories, Legacy Research, 1225 NE 2nd Ave-
nue, Portland, OR 97232, USA.
E-mail: JSaugstad@DowNeurobiology.org
This study was supported by National Institutes of Health NINDS
R21 NS054220 (JAS).
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
&
2010 ISCBFM All rights reserved 0271-678X/10
$32.00
www.jcbfm.com
Page 1
translating ribosomes and protein synthesis (Bhatta-
charyya et al, 2006). MiRNAs expressed within
dendrites regulate translation of proteins mediating
dendritic growth (Schratt et al, 2006). MiRNAs that
regulate synaptic plasticity can target, and be
targeted by, plasticity mediators such as cAM P
response element binding protein (CREB), fragile X
mental retardation protein, and MeCP2 (methyl-CpG
binding protein 2) (Smalheiser and Lugli, 2009).
We propose that ischemic preco nditioning could
regulate miRNA expression and thus serve as novel
effectors of altered protein expression that leads to
ischemic tolerance. Accordingly, we show that
ischemia does result in significant changes in
miRNA express ion in preconditioned, ischemic,
and tolerant cortices, relative to sham. Target
prediction analysis revealed MeCP2 as the most
prominent target of miRNAs regulated in precondi-
tioned cortex; thus, we further propose that the
preconditioning-regulated miRNAs serve, at least in
part, to regulate protein expression of transcriptional
regulators required for ischemic tolerance.
MeCP2 has been considered as a global transcrip-
tional repressor because of its methyl-DNA binding
and transcription repression domains. For example,
in neurons, MeCP2 bound to the brain-derived
neurotrophic factor promoter is released on mem-
brane depolarization, resulting in transcription of
brain-derived neurotrophic factor mRNA (Chen et al,
2003). However, results of new studies indicate that
MeCP2 is a global regulator of transcription; MeCP2
can repress transcription when complexed with
histone deacetyl ase, or activate transcription when
complexed with CREB1 (Chahrour et al, 2008).
Further studies have established MeCP2 as a multi-
functional nuclear protein with roles in chromatin
architecture, regulation of RNA splicing, and tran-
scriptional activation (Hite et al, 2009). Together,
these studies show a complex role for MeCP2 in
brain function and synaptic plasticity; as such,
MeCP2 is associated with epigenetic regulation of
the nervous system (MacDonald and Roskams, 2009).
The results of our studies show that precondition-
ing regulates miRNA expression, and target predic-
tion of the preconditioning-regulated miRNAs
identified novel proteins that could serve as effectors
governing the epigenetic changes that mediate pre-
conditioning-induced tolerance. Among these novel
proteins, expression of MeCP2 protein increases in
preconditioned cortex, with no corresponding
change in MeCP2 mRNA ex pression, consistent with
speculation that MeCP2 is posttranscriptionally
regulated (Shahbazian et al, 2002). Accordingly,
MeCP2 protein is directly regulated by miR-132 in
neurons: decreased miR-132 increases MeCP2 ex-
pression (Klein et al, 2007). Although neither
miRNAs nor MeCP2 have previou sly been linked to
ischemic preconditioning, these studies support the
concept that both could serve as novel effectors of
the molecular mechanisms underlying ischemic
preconditioning-induced tolerance.
Materials and methods
Transient Focal Ischemia
Adult male C57BL/6J mice (25 to 30 g; Charles River
Laboratories, Wilmington, MA, USA) were maintained
under diurnal conditions (12 h light/dark cycle) on an ad
libitum lab chow and drinking water. Experiments were
performed in accordance with the Association for Assess-
ment and Accreditation of Laboratory Animal Care and
approved by the Institutional Animal Care and Use
Committee of Legacy Research. Transient focal ischemia
was induced by suture occlusion of the middle cerebral
artery (MCAO) in male mice anesthetized using 1.5%
isoflurane, 70% N
2
O, and 28.5% O
2
(Longa et al, 1989).
Cerebral blood flow in the middle cerebral artery was
monitored through a fiber optic probe, mice showing less
than 70% cerebral blood flow reduction were excluded
from the analysis. Mouse brains were removed 24 h after
final MCAO and the ipsilateral and contralateral cortices
were dissected and frozen at 801C. Three brains from
each group were stained with vital dye TTC (2,3,5-
triphenyltetrazolium hydrochloride), sliced into six sec-
tions, and each section was scanned. The stained and
unstained areas of each hemisphere were quantified with
ImageJ 1.32j (NIH, Bethesda, MD, USA), and values were
used to calculate infarct volume expressed as a percentage
of the unlesioned hemisphere.
MicroRNA Microarray
RNA was isolated from each cortex using the mirVana
miRNA Isolation Kit (Ambion, Austin, TX, USA). The
Duke University Microarray Facility labeled RNAs from
the ipsilateral (treated) cortex with Cy5 fluorophore, and
RNAs from the contralateral (untreated) cortex with Cy3
fluorophore (Amersham Biosciences, Piscataway, NJ,
USA). Labeled RNAs were hybridized with mirVana Probe
Set V2 (Ambion) microarray slides including probes for
human (328), mouse (115), and rat (46) miRNAs. The
microarray slides were scanned on a GenePix 4000B (Axon
Instruments, Union City, CA, USA). Total Cy3 signal was
set to equal total Cy5 signal for each microarray, thus the
ratio of total Cy3 to total Cy5 was 1.
MicroRNA Microarray Data Analysis
The miRNA microarray data were analyzed using a Web-
based miRNA microarray analysis program created by Rob
Lusardi (Slowdog Software, Portland, OR, USA). The ratio
of the median intensities for each signal was calculated: 1
indicated equal quantities of target miRNA in the ipsilat-
eral and contralateral cortices, < 1 reduced quantities of
target miRNA in the ipsilateral cortex, and > 1 increased
quantities of target miRNA in the ipsilateral cortex, relative
to contralateral cortex. Each ratio was log
2
-transformed to
produce a normally distributed data set amenable to
standard statistical analysis: average log ratio (ALR) of
miRNA expression = log
2
(Cy5/Cy3), where Cy5 and Cy3
were the probe intensities of a single miRNA in the
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
745
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 2
ipsilateral and contralateral cortices, respectively, from the
same mouse. A log ratio of 0 indicated no change between
ipsilateral and contralateral cortices, positive values
indicated increased miRNA expression in ipsilateral
cortex, and negative values decreased expression in
ipsilateral cortex, relative to contralateral cortex. The
ALR of miRNA expression in preconditioned, ischemic,
or tolerant cortex was compared with that of miRNAs in
sham cortex, and Student’s t-test was used to identify those
miRNAs that were statistically different from sham.
Target prediction was limited to high-confidence
miRNAs, defined as those present in all experimental
replicates. Analysis was performed using miRanda (ver-
sion 2005; www.microrna.org) (John et al, 2004) that allows
queries of several miRNAs and reports the total number of
gene targets (ENSG), mRNA transcripts (ENST), and the
number of ‘hits’ that are predicted sites for miRNA binding
based on mRNA complementarity (miRNA/ENST pair).
The mRNA binding sites are located in the 3
0
UTR,
numbered 5
0
to 3
0
from nucleotide 1 beginning just after
the stop codon.
Quantitative Real-Time Polymerase Chain Reaction
We analyzed miRNA expression in mouse cortex by qRT-
PCR using the miRCURY RNA miRNA PCR System with
miR-132 and control U6 primer sets labeled with SYBR
Green (Exiqon Inc., Woburn, MA, USA). The mRNA qRT-
PCRs were performed using primer sets for MeCP2,
SLC2A3, and 18S RNA with TaqMan FAM-labeled probe
reagents on a 7500 Fast Real-Time PCR System (Applied
Biosystems Inc., Foster City, CA, USA).
MeCP2 Knockout Mice
MeCP2 knockout (KO) mice (strain B6.129P2(C)-
Mecp2
tm1.1Bird
/J) were obtained from Jackson Laborato-
ries (Bar Harbor, ME, USA). Heterozygous females
(Mecp2
tm1.1Bird
/J) crossed with wild-type (WT) males
(C57BL/6J) yielded WT females (Mecp2
+
/Mecp2
+
), hetero-
zygous females (Mecp2
/Mecp2
+
), WT males (Mecp2
+
/y),
and KO males (Mecp2
/y) (Guy et al, 2001). Genotyping
was confirmed by PCR using DNA isolated from tail
biopsies, as per the Jackson Laboratories protocol.
Immunoblots
Nuclear fractionation of mouse brain cortices was per-
formed using the CelLytic NuCLEAR Extraction Kit (Sigma,
St Louis, MO, USA). Proteins separated by one-dimensional
gel electrophoresis were transferred to polyvinylidene
difluoride membrane (Millipore, Billerica, MA, USA).
Protein blots were incubated in 1:1,000 dilution of mouse
antihuman MeCP2 antibody (ab50005; Abcam, Cambridge,
MA, USA) then incubated in 1:10,000 dilution of goat
antimouse horseradish-peroxidase-conjugated secondary
antibody (Bio-Rad Laboratories, Hercules, CA, USA). Pro-
tein bands were detected using enhanced chemilumines-
cence (Amersham Biosciences) and Kodak BioMax film
(Eastman Kodak Co., Rochester, NY, USA). Blots were
scanned and quantified using the Kodak Image Station
2000rt and Kodak 1D version 3.6 software. Protein bands
were background-subtracted, normalized to a-tubulin III
(46 kDa; Sigma T8660), and the normalized intensity was
expressed as relative to the average control tissue. Statistical
analysis was performed by analysis of variance (ANOVA),
with a post hoc Dunnett test for significant differences.
Immunohistochemistry
Mouse brains were flash frozen and sectioned on a Cryostat
CM3050S (Leica Microsystems Inc., Bannockburn, IL,
USA) into 12-mm-thick sections. Whole-brain mounts were
fixed, permeabilized, blocked, and incubated with rabbit
antimouse MeCP2 antibody (07-013; Millipore/Upstate),
then incubated with goat antirabbit Cy3-conjugated anti-
body (111-165-003; Jackson ImmunoResearch Laboratories,
West Grove, PA, USA). Slides were mounted with
Vectasheild (Vector Labs, Burlingame, CA, USA) contain-
ing DAPI (4
0
,6-diamidino-2-phenylindole) and images were
captured on a Leica TCS SP2 microscope (Leica Micro-
systems Inc.). For Nissl stain, whole-brain mounts were
incubated in cresyl violet, covered in permount, and
coverslipped. Each slice was scanned, and stained and
unstained areas of the treated hemisphere were quantified
using ImageJ 1.32j (NIH) to calculate the infarct volume as
a percentage of the unlesioned hemisphere.
Results
Ischemic Preconditioning Alters miRNA Expression in
Adult Mouse Cortex
We generated preconditioned, ischemic, and tolerant
mice using varying durations of MCAO (Figure 1A).
Representative images of TTC-stained sections reveal
the extent of injury in mouse brains (Figure 1B).
Infarct volume was quantified using TTC-stained
sections (n = 3 mice per group). Sham (S) mice
show no injury, whereas preconditioned (P) mice
show injury in striatum, and ischemic (I) mice show
significant injury in cortex and striatum. Prior pre-
conditioning provides tolerance (T) against ischemic
challenge in cortex evident by 24 h (T-24), more
robust at 72 h (T-72), and gone by 10 days after pre-
conditioning (T-240). Representative miRNA micro-
array heatmaps show consistent patterns of miRNA
expression within sham (n = 3) and preconditioned
(n = 3) mice (Figure 1C), and distinct patterns between
sham and preconditioned treatments. Thus, we ex-
amined sham, preconditioned, ischemic, and tolerant
mouse cortices by miRNA microarray analysis.
Statistical Analysis Reveals Distinct Changes in
miRNA Expression in Preconditioned, Ischemic,
and Tolerant Mouse Cortices
We first evaluated the consistency of miRNA expres-
sion within each group to identify animal-to-animal
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
746
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 3
and/or diurnal variations in miRNA expression . For
each mouse, the ALR of miRNA expression from one
animal (Replicate) was compared with the ALR of
miRNA expression for all replicates in the same
group (Total); each point in Figure 2A represents one
unique miRNA. Represent ative graphs show a posi-
tive correlation between one individual replicate
relative to the average of the total replicates sampled
within each group for sham (Figure 2Ai), precondi-
tioned (Figure 2Aii), ischemic (Figure 2Aiii), and
tolerant (Figure 2Aiv) animals. These studies show
that miRNA expression is consistent within a
treatment group and that regulation of miRNAs is
not random in individual mice.
We then analyzed miRNA expression in the
ipsilateral cortex of mice in each group, relative to
contralateral cortex (Figure 2B). A histogram of the
ALR for sham miRNAs (n = 9) shows a normal
distribution near zero, with a mode of 0.20,
indicating that miRNA expression between the
cortices is similar (Figure 2Bi). However, a histogram
of the ALR for miRN As in preconditioned cortex
(n = 6) shows a broadening of the distribution with
a negative ‘tail’ region, with a shift in the mode to
+ 0.28, indicating that miRNA expression is both
increased and decreased (P < 0.05, t-test; P < 0.001,
F test; Figure 2Bii). In contrast, a histogram of the
ALR for miRNAs in ischemic cortex (n = 6) shows a
broadening of the distribution with a negative ‘tail’
region, but a shift in the mode to 0.14 is not large,
indicating a bias toward decreased miRNA expres-
sion (P < 0.001, t-test; P < 0.001, F test; Figure 2Biii).
However, a histogram of the ALR for miRNAs in
tolerant cortex (n = 3) shows a tighter, normal
distribution and a negative shift in the mode to
0.38, similar to distribution in the sham group
(P < 0.001, t-test; P < 0.001, F test; Figure 2Biv). These
studies show that each treatment induced distinct
changes in mouse cortical miRNA expression.
We then compared miRNA exp ression in the
preconditioned, ischemic, and tolerant mice to those
of the sham mice (Figure 2C). Graphs show the total
ALR for each miRNA in a group plotted against the
total ALR of miRNAs in sham. Sham versus sham is
provided to illustrate the expected outcome of a
positive correlation in unchanged miRNA expression
(Figure 2Ci). The graphs show that the ALRs of
miRNAs from preconditioned cortices are negatively
correlated to sham values (Figure 2Cii), and a similar
negative correlation is seen in the ALR of miRNAs
from ischemic cortices (Figure 2Ciii). In contrast,
there is a positive correlation between the ALR of
miRNAs from tolerant cortices relative to sham
Injury in Mouse MCAO Models
Sham
Mouse Cortex MicroRNA Microarrays
Ipsilateral
(Treated)
Contralateral
(Untreated)
C
T
X
ST
60
0
10
20
30
40
50
% Infarct volume
*
*
T240T24 T72SP I
1) Preconditioned
15 min
MCAO
2) Ischemic
3) Tolerant
Reperfusion (hrs)
15 min
MCAO
60 min
MCAO
60 min
MCAO 24 hr
24 hr
24 hr
Mouse Ischemia Models
Preconditioned
Figure 1 Ischemic preconditioning and microRNA microarrays
in adult mouse cortex. ( A) Schematic of MCAOs used to generate
preconditioned (15 mins), ischemic (60 mins), or tolerant
(15 mins, reperfusion, 60 mins) mouse brains, all reperfused
24 h after final MCAO. (B) Images of TTC-stained mouse brains
revealing injury in each group, and a graph showing percent
infarct volume for each condition (n = 3 per group). Sham (S)
mice are uninjured, preconditioned (P) show little injury in
ipsilateral cortex, ischemic (I) show significant injury in
ipsilateral cortex and striatum, tolerant (T) show robust
protection of ipsilateral cortex against ischemic injury that is
apparent by 24 h (T-24), more robust 72 h (T-72) and gone 10
days (T-240) after preconditioning. (C) Schematic of ipsilateral
cortex (CTX) and striatum (ST) targeted by MCAO. Representa-
tive heatmaps show miRNAs from preconditioned mice both
increase (red) and decrease (blue) signal intensities, relative to
sham mice.
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
747
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 4
(Figure 2Civ). These results show distinct changes in
miRNAs in the ipsilateral cortex of preconditioned,
ischemic, and tolerant cortices, relative to sham.
Individual miRNAs from preconditioned, ischemic,
and tolerant cortices were then examined by t-test to
identify stat istically significant changes in expres-
sion, relative to sham. A Venn diagram (Figure 2D)
illustrates that of 488 unique miRNAs in the
microarray probe set, 273 miRNAs in preconditioned
(P), 144 miRNAs in ischemic (I), and 50 miRNAs
in tolerant (T) cortex were significantly different
(P < 0.05) from miRNAs in sham. The 31 miRNAs
regulated in all treatment groups likely represent
miRNAs altered in response to stress. MiRNAs in
preconditioned cortex increased and decreased
(Figure 2E, All P), where as miRNAs in ischemic
and tolerant cortices predominantly decreased
(Figure 2E, All I, T). Of miRNAs uniq uely regu-
i. Sham #3
Total Sham
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
3
2
1
-3
-2
-1
Total Preconditioned
-1
ii. Preconditioned #6
Total Ischemic
iii. Ischemic #1
Total Tolerant
iv. Tolerant #1
ALR
Mode = -0.38
SD = 0.25
iv. Tolerant
Mode = -0.14
SD = 0.50
iii. Ischemic
Mode = 0.28
SD = 0.77
ii. Preconditioned
# of MicroRNAs
Mode = -0.20
SD = 0.18
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
10
20
30
40
50
60
i. Sham
i. Total Sham
Total Sham
Total Sham
ii. Total Preconditioned
Total Sham
iii. Total Ischemic
Total Sham
iv. Total Tolerant
3
86
13
31
I
26
P
157
T
3
200
-150
-100
-50
0
50
100
150
All Common
# of MicroRNAs
321-3 -1-2
321-3
-1
-2 321-3
-1
-2 321-3
-1
-2 321-3
-1
-2
321-3 -1-2 321-3 -1-2 321-3 -1-2
-2-3
3210-1
ALR
-2-3
3210-1
ALR
-2-3 3210-1
ALR
-2-3 3210-1
IPPITPTITPITIPT
Unique
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
748
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 5
lated in preconditioned cortex, 153 increased and
4 decreased (Figure 2E, Unique P), and of miRNAs
uniquely regulated in ischemic cortex, 2 increased
and 24 decreased (Figure 2E, Unique I). Further,
of miRNAs uniquely regulated in tolerant cortex,
one increased and two decreased (Figure 2E,
Unique T). Remaining data reflect miRNAs regulated
among two or more groups (Figure 2E, Common).
These results show that miRNA expression is
significantly regulated in the ipsilateral cortex in
preconditioned, ischemic, and tolerant mice. Sup-
plementary Tables 1–3 list those miRNAs signifi-
cantly regulated in preconditioned, ischemic, or
tolerant mouse cortices.
Target Prediction of the miRNAs Regulated in
Preconditioned Mouse Cortex
We used bioinformatic software tools to identify
potential mRNA targets of the miRNAs significantly
regulated in preconditioned mouse cortex (John et al,
2004), and restricted our an alysis to high-confidence
miRNAs, defined as those miRNAs detected in all
microarray replicates (nine sham, six precondi-
tioned, six ischem ic, three tolerant). Given the
cooperative nature of miRNAs and that target
regulation is more potent when several miRNAs
bind to a given mRNA (Doench and Sharp, 2004), the
number of ‘hits,’ or miRNA binding sites on an
mRNA 3
0
UTR is an important consideration in target
prediction. Thus, we used miRanda (version 2005)
for target prediction as it can query several miRNAs
at one time to identify predicted mRNA targets of a
cohort of miRNAs. Significantly regulated (P < 0.05)
high-confidence miRNAs included 205 miRNAs; of
these, 152 miRNAs were present in miRanda. The
queries revealed that the number of predicted targets
exceeded 28,000 unique Ensemble gene ID mRNAs,
and given that many mRNAs have multiple miRNA
binding sites, also revealed over 130,000 potential
miRNA to mRNA interactions (Supplementary Table
4). Sorting the Ensemble gene ID targets of miRNAs
in preconditioned cortex by the number of potential
hits revealed that a striking number of transcriptional
regulators are heavily targeted by the miRNAs.
Assessment of Gene Ontology of the precondition-
ing-regulated miRNA targets using Fatigo (Al-Shah-
rour et al, 2005; www.babelomics.org/, accessed on 7
December 2007) confirmed that the most prominent
group of mRNA targets encode proteins that function
as regulators of transcription (Table 1).
The mRNA predicted to be most heavily targeted
by the preconditioning-regulated miRNAs encodes
for MeCP2. DNA microarray studies show that
MeCP2 mRNA
is not significantly regulated by
preconditioning (Stenzel-Poore et al, 2003), suggest-
ing that protein expression is regulated at the
posttranscriptional level. Given that MeCP2 had not
previously been examined in the context of precon-
ditioning, we focused further studies on this protein.
We first analyzed the nature and distribution of
miRNAs predicted to bind to MeCP2 mRNA. The
MeCP2 mRNA transcripts (NM_001081979,
NM_010788) are identical throughout their 8,596
nucleotide 3
0
UTR, and of 76 miRNAs predicted to
target 117 sites in the 3
0
UTR (miRanda, version
2005), 58 miRNAs targeting 92 total sites are
Figure 2 Distinct changes in microRNA expression in preconditioned, ischemic, and tolerant mouse cortices. (A) miRNA expression
patterns are consistent within each group, and ALR of miRNA expression in each cortex was compared with average ALR of miRNA
expression for all replicates in the same group (Total). Representative correlations are shown for (i) sham #3, (ii) preconditioned #6,
(iii) ischemic #1, and (iv) tolerant #1. (B) Histograms of miRNA distribution for sham (i) mice show ALR values near zero,
preconditioned (ii) and ischemic (iii) mice both show large populations of decreased miRNAs, whereas preconditioned mice also
show a large number of increased miRNAs. Tolerant (iv) mice ALRs are similar to sham with a shift toward decreased expression in
the ipsilateral cortex. (C) Differences in miRNA expression in each group, relative to sham. Average total response for each group is
plotted against the average total response in sham; each point represents the average ALR for a unique miRNA. Sham versus sham
(i) illustrates a positive correlation in unchanged miRNA expression. Preconditioned (ii) and ischemic (iii) reveal negative
correlations, in contrast to a positive correlation in tolerant (iv), relevant to sham. (D) A Venn diagram illustrating 287 miRNAs
significantly altered in preconditioned (P) cortex relative to sham: 157 are uniquely altered, 86 in common with ischemic (I), 13 in
common with tolerant (T), and 31 in common in all three groups. (E ) Graph of miRNA distribution in each group shows number of
miRNAs significantly increased or decreased in preconditioned (P), ischemic (I), or tolerant (T) cortex for All (total miRNAs), Unique,
or Common (two or more groups).
Table 1 Prominent messenger RNA targets of high-confidence
preconditioning-regulated microRNAs (total number of unique
miRNAs targeting the 3
0
UTR/number of potential miRNA/mRNA
binding sites)
Protein Of all miRNAs represented
on the microarrays
Preconditioned miRNAs
*+
MeCP2 61/96 18/35 31/44
HDAC4 30/41 9/11 16/26
CREB1 11/22 4/10 4/8
BDNF 27/136 8/52 16/72
MEF2C 18/57 7/24 7/21
DICER1 17/21 5/8 7/8
EIF2C2 15/15 1/1 11/11
CAMK2A 23/33 8/10 11/18
Abbreviations: MeCP2, methyl-CpG binding protein 2; HDAC4, histone
deacetylase 4; CREB1, cAMP response element binding protein;
BDNF, brain-derived neurotrophic factor; MEF2C, myocyte-specific
enhancer factor 2C; DICER1, endoribonuclease dicer; EIF2C2, eukaryotic
translation initiation factor 2C 2; CAMK2A, Ca
2+
/calmodulin-dependent
protein kinase II a.
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
749
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 6
significantly regulated in preconditioned cortex
(Table 2A). In contrast, glucose transporter 3
(SLC2A3) mRNA
is significantly increased in pre-
conditioned mouse cortex (Stenzel-Poore et al, 2003).
SLC2A3 mRNA transcript (NM_011401) contains a
3,716 nucleotide 3
0
UTR, and of 13 miRNAs
predicted to target 13 sites in the 3
0
UTR, 10 miRNAs
targeting 11 total sites are significantly altered in
preconditioned cortex (Table 2B). These predictions
support that MeCP2 mRNA is a prominent target of
the preconditioning-regulated miRNAs.
Ischemic Preconditioning Decreases miR-132 and
Increases MeCP2 Protein, but has no Effect on
MeCP2 mRNA Levels
Those miRNAs predicted by miRanda to target the
MeCP2 3
0
UTR are depicted in Figure 3A; among the
decreased miRNAs is miR-132. Klein et al (2007)
have shown that MeCP2 expression is controlled by
miR-132: decreased miR-132 leads to increased MeCP2
expression in neurons, whereas increased miR-132
leads to decreased MeCP2 expression. Results of our
miRNA microarray data show that miR-132 expres-
sion is significantly decreased in preconditioned (P)
cortex (Figure 3B, 2.26
±
0.485 ALR, n = 6), relative
to control (C). Consistent with this, we also show
qRT-PCR data validating decreased expression of
miR-132 at 24 h after preconditioning (P24) (Figure 3C,
0.75
±
0.243 DDC
t
, n = 4), relative to control (C).
These studies validate that miR-132, an miRNA
known to regulate MeCP2 protein expression, is
decreased in preconditioned mouse cortex.
We then used immunoblot analysis to quantify
MeCP2 protein expression in preconditioned mouse
cortex. A represen tative immunoblot shows MeCP2
expression in control (C) and precondition ed (P)
Table 2 Preconditioning-regulated microRNAs predicted to the target (A) MeCP2 mRNA 3
0
UTR and (B) SLC2A3 mRNA 3
0
UTR
Decreased mRNA target site(s) Increased mRNA target site(s)
A
miR-22 29 miR-30e-3p 77, 8373
miR-425 69 miR-302b-AS 193
miR-218 183 miR-378 195, 4850
miR-331 204, 4134, 4487, 6075 miR-19a 224, 961
miR-149 207, 4138, 4264, 7519 miR-301 226, 964
miR-328 209 miR-424 263, 264, 1161, 1162, 2159, 2160
miR-222 213 miR-199a-AS 267
miR-106a 217 miR-17-3p 268, 7989
miR-17-5p 217 miR-183 379, 3665
miR-370 828, 3113, 4113, 5964 miR-346 784, 3126, 5729
miR-339 879, 3195, 3807, 6068 miR-196b 1067
miR-145 1013, 6719 miR-296 1643, 6077, 6098
miR-103 2156 miR-147 1871
miR-15a 2157 miR-340 2101
miR-15b 2159 miR-337 2152
miR-16 2160 miR-373 2264
miR-138 3241 miR-302b 2267
miR-320 3348 miR-302d 2267, 8380
miR-29a 3359 miR-372 2268, 8381
let-7c 3428 miR-96 3666
let-7b 3428 miR-34b 4483
let-7a 3430, 4966 miR-214 4912
let-7d 3431 miR-200c 5271
let-7e 3431, 7959 miR-141 5272
miR-379 3480 miR-330 5351
miR-185 4400
let-7f 4477
miR-34a 4481
miR-24 4936, 6456, 6889
miR-133a 6074
miR-132 6870
miR-212 6875
miR-27b 7752
B
miR-103 67 miR-140 22
miR-107 73 miR-424 80, 81
miR-15b 76 miR-338 671
miR-16 79 miR-148b 1465
miR-15a 80
miR-195 80
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
750
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 7
mouse cortical nuclear lysates (Figure 3C). The
MeCP2 antibody (ab50005; Abcam) detects more
than one protein band on the immunoblots, thus
we also examined nuclear lysates from MeCP2 KO
mice to confirm that the 72 kDa protein band (white
diamond) is indeed MeCP2. a-Tubulin III (T), which
served as a loading control, is shown in the bottom
panel. Quantitative analysis of MeCP2 protein
expression for control (C, n = 3) and preconditioned
(P, n = 3) mice reveals a significant increase in MeCP2
protein in preconditioned cortex (P < 0.05 by t-test)
relative to control. Thes e studies reveal the novel
finding that ischemic preconditioning increases
protein expression of MeCP2, the most prominent
predicted target of the preconditioning-regulated
miRNAs.
DNA microarray studies do not find MeCP2 mRNA
regulated in preconditioned cortex (Stenzel-Poore
et al, 2003). Thus, we quantified expression of MeCP2
mRNA by qRT-PCR at 8, 24, 48, and 72 h after
preconditioning (n = 3 mice per time point, including
sham). The results show no significant change in
MeCP2 mRNA any time after preconditioning rela-
tive to sham (Figure 3E; DC
t
= shamexperiment),
and repeated-measures ANOVA confirmed that
preconditioning does not affect MeCP2 mRNA
(P < 0.68). DNA microarray shows increased SLC2A3
mRNA in preconditioned mouse cortex (Stenzel-
Predicted miRNA Binding Sites on MeCP2 3’UTR
77
193
195
224
226
263
267
268
379
784
961
964
1067
1162
1161
1643
1871
2101
2152
2159
2160
2264
2267
2268
3126
3665
3666
4483
4850
4912
5271
5272
5351
5729
6077
6098
8373
8380
8381
29
69
183
204
207
209
213
217
828
879
1013
2157
2159
2160
3113
3195
3241
3348
3359
3428
3430
3431
3480
3807
4477
4481
4487
4936
4966
4113
4138
4264
4400
4134
5964
6074
6075
6456
6870
6875
6889
6719
7519
7752
7959
5’
3’
Predicted SLC2A3 3’ UTR
miRNA Binding Sites
5’
3’
67
73
76
79
80
80
22
80
81
671
1465
-1.0
-0.5
0.0
0.5
1.0
1.5
ΔCt (± SE)
MeCP2 mRNA
P8 P24 P48 P72
S
SLC2A3 mRNA
*
P24 P48 P72P8S
-1.0
-0.5
0.0
0.5
1.0
1.5
ΔCt (±SE)
MeCP2 Protein
C
250
150
100
50
75
T
Normalized Intensity
(mean ± SE)
0.0
1.0
2.0
3.0
4.0
5.0
*
MeCP2 Protein
miR-132
P24S
ALR (± SE)
-3.0
-2.0
-1.0
0
*
-1.2
-0.8
-0.4
0
ΔΔCt (± SE)
P48
P24P12
P6C
miR-132
*
2156
6068
7989
KOP
P
C
KO
Figure 3 Decreased miR-132 expression correlates with increased MeCP2 protein, but no change in MeCP2 mRNA, in
preconditioned mouse cortex. (A) Predicted MeCP2 3
0
UTR target sites for increased (up arrow) and decreased (down arrow) miRNAs
significantly regulated in preconditioned cortex. (B) MiR-132 expression by microarray shows decreased ALR of 2.23
±
0.485
(n = 6) in preconditioned (P) cortex, relative to sham (S) (0.026
±
0.211 ALR, n = 9). (C) MiR-132 expression by qRT-PCR
validating decreased miR-132 expression (n = 4) 24 h after preconditioning (P24, 0.75
±
0.34 DDC
t
), relative to control (C). (D)
Immunoblot of mouse cortex showing MeCP2 protein (72 kDa, white diamond) significantly increased in preconditioned (P) cortex,
relative to control (C). a-Tubulin III (T) served as a loading control (bottom panel). Quantification of control (C) and preconditioned (P)
protein bands (n = 3 each) validates a significant increase in MeCP2 protein (P < 0.05). (E) MeCP2 mRNA expression at 8, 24, 48,
and 72 h after ischemic preconditioning and in sham cortex expressed as DC
t
(shampreconditioned) shows no detectable changes
in MeCP2 mRNA in preconditioned cortex at the time point (data analyzed by repeated measures ANOVA). (F) Schematic of
predicted SLC2A3 target sites for increased (up arrow) and decreased (down arrow) miRNAs significantly regulated in preconditioned
cortex. (G) SLC2A3 mRNA expression expressed as DC
t
(sham-preconditioned) shows a significant increase in preconditioned cortex
(P < 0.05 by repeated measures ANOVA).
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
751
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 8
Poore et al, 2003) and the SLC2A3 3
0
UTR is not
a prominent target of preconditioning-regulated
miRNAs (Figure 3F). Accordingly, SLC2A3 mRNA
levels are significantly increased in preconditioned
cortex (Figure 3G, n = 3 mice per time point) relative
to sham (DC
t
= shamexperiment) with P < 0.05 by
repeated-measures ANOVA. Fisher post hoc test
revealed a significant difference between sham and
8 h preconditioning (P < 0.05). These studies validate
that ischemic preconditioning has no effect on MeCP2
mRNA but does induce a significant increase in
SLC2A3 mRNA.
MeCP2 Knockout Mice Show Increased Susceptibility
to Preconditioning Ischemia
We next examined the cellular distribution and
temporal expression of MeCP2 protein in control,
sham, and at 8, 24, 48, and 72 h after preconditioning
by immunohistochemistry (n = 3 mice per time
point). Representative images show increased
MeCP2 protein in mouse cortical cells by 8 h after
preconditioning, persisting at 24 h but not apparent
by 48 h after preconditioning (Figure 4A, Cortex). In
contrast, MeCP2 protein does not change in the
striatum, a region of the brain that is not protected by
preconditioning (Figure 4A, Striatum). Given the
restricted expression of MeCP2 in the protected
cortex, we examined whether depletion of MeCP2
would affect the ability of mice to form ischemic
tolerance. Thus, we examined the ability of MeCP2
KO mice to form ischemic preconditioning-induced
tolerance. The KO mice had a high mortality rate in
response to the ischemic challenge administered 72 h
after preconditioning, and only one of three KO mice
survived. However, Nissl stain from the surviving
KO mouse shows extensive injury in the cortex,
relative to the WT mice (Figure 4B, Tolerance). We
then examined the effect of preconditioning ische-
mia alone on KO mouse brains. Representative Nissl-
stained brain sections from preconditioned WT mice
with a 72 h recovery (Figure 4B) show no injur y in
the cortex, and neuronal loss in the striatum (n = 3).
Further, there is no apparent injury in the cortex of
the preconditioned KO mice (n = 3). However, higher
magnification of the brain sections revealed irregular,
nonuniformly stained nuclei in the cortex of KO
mice, in contrast to cells in the cortex of WT mice
(n = 3 each). Calculated infarct volumes of WT mice
from the Nissl-stained sections are similar to the
TTC-stained sections, showing the same resul t from
both techniques (Figure 4C). The Nissl-stained sec-
tions show comparable infarct volumes in precondi-
tioned (P) WT and KO mice (n = 3 each). However, the
tolerant (T) KO mouse that survived the ischemic
challenge (one of three) had extensive injury in the
cortex, relative to WT mice. These results show that
KO mice have an increased susceptibility to ischemia,
and that further studies to examine a role for MeCP2
and other transcriptional regulators as effectors of
ischemic tolerance are warranted.
Discussion
Cerebral miRNA expression is regulated by transient
ischemia (Dharap et al, 2009; Jeyaseelan et al, 2008),
traumatic brain injury (Redell et al, 2009), and
several other neurologic disorders (Kuss and Chen,
2008). Ischemic preconditioning-induced tolerance
requires de novo protein synthesis (Barone et al,
1998), is correlated with genomic reprogramming of
the brains response to ischemia (Bowen et al, 2006;
Koerner et al, 2007; Stenzel-Poore et al, 2003), and
tolerance induced by prior preconditioning is tran-
sient (Chen et al, 1996; Perez-Pinzon et al, 1997). Our
studies herein focus on miRNAs as effectors of
ischemic preconditioning-induced tolerance based
on their role as regulators of posttranscriptional gene
expression (Bartel, 2004; Chen and Meister, 2005;
Smalheiser and Lugli, 2009). We used microarrays
to analyze miRN A expression in preconditioned,
ischemic, and tolerant mouse cortices. We show that
miRNA expression is consistent among mice within
a treatment group, miRNA distribution profiles
reflect specific responses to each treatment, and
miRNA expression profiles in preconditioned,
ischemic, and tolerant mouse cortices are signifi-
cantly different (P < 0.05) from sham-operated mice.
As preconditioning was the foremost regulator of
miRNAs, we focused our bioinformatic studies on
identifying potential mRNA targets of the precondi-
tioning-regulated miRNAs. As repressed mRNA
translation is enhanced for those mRNAs targeted
by multiple miRNAs (Doench and Sharp, 2004), we
used miRanda (version 2005) for target prediction as
this program allows simultaneous queries of multi-
ple miRNAs. Using t-tests to determine significant
changes in treated mice, we identified hundreds
of miRNAs as potentially significant, even though
their fold changes were frequently small. The power
of miRanda prediction software is that we were
able to predict the cumulative effects of many small,
yet significant, miRNA changes on protein expres-
sion. The prediction studies revealed that the
most prominent targets of preconditioning-regulated
miRNAs were transcriptional and translational reg-
ulators, consistent with reports that overrepresente d
groups of miRNA targets include transcription
factors, components of the miRNA machinery, and
proteins involved in translational regulation (John
et al, 2004). As MeCP2 mRNA was the most promi-
nent target of preconditioning-regulated miRNAs, we
selected it for further study and show that ischemic
preconditioning rapidly increased MeCP2 protein,
but not MeCP2 mRNA, in mouse cortex. These
results are consistent with the suggestion that MeCP2
translation might be posttranscriptionally regulated
by tissue-specific factors (Shahbazian et al, 2002).
We examined MeCP2 protein because as a tran-
scriptional repressor (Fuks et al, 2003), MeCP2 could
be an effector of gen omic repro gramming, and had
not been examined in the context of preconditioning.
Thus our finding that MeCP2 expression is rapidly
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
752
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 9
increased in preconditioned cortex provided a
potential link between two key, yet seemingly
paradoxical, features of ischemic preconditioning-
induced tolerance: the requirement for new protein
synthesis (Barone et al, 1998) and genomic repro-
gramming of the response to ischemia that leads to a
transient repression of gene expression (Stenzel-
Poore et al, 2003). MeCP2 is a potent transcriptional
repressor, yet recent studies show that MeCP2 is a
complex regulator of transcription (Chahrour et al,
2008): MeCP2 as a transcriptional activator requires
CREB1, and as a transcriptional repressor requires
histone deacetyl ase. Since MeCP2 had not pre-
viously been examined in the context of precondi-
tioning or tolerance, it also served as a novel
protein to test the power of miRNA target prediction.
These studies support the concept that, in addi-
tion to preconditioning-indu ced changes in gene
MeCP2 Protein
CortexStriatum
Preconditioned
Tolerant
WT Cortex StriatumCortex
Cortex StriatumCortex
WT
MeCP2 KO MeCP2 KO
Infarct Volume
60
0
10
20
30
40
50
% Infarct Volume
KO
NisslTTC
WT
PPT PT
T
SC P72P48
P24P8
Figure 4 Increased susceptibility to ischemia in MeCP2 KO mouse cortex. (A) MeCP2 protein expression increased in cortex by 8 h
(P8) after preconditioning, maintained at 24 h (P24), and returned to control levels by 48 h (P48, P72) after preconditioning.
MeCP2 protein is not increased in preconditioned striatum any time after preconditioning. Scale bar: 100 mm. (B) Nissl-stained WT
and KO mouse brains subjected to tolerance or preconditioning (72 h recovery) show extensive cell death in the ipsilateral cortex of a
tolerant KO mouse (n = 1), but not in the cortex of WT mice (n = 3). Preconditioned mice show no apparent injury in the cortex of
preconditioned WT or KO mice (n = 3 each). However, magnified images reveal injured cortical cells in preconditioned KO, but not
WT, mice (n = 3 each). Cells in the striatum of WT and KO mice are injured by preconditioning. Scale bar: 50 mm. (C) Percent infarct
volume for TTC- and Nissl-stained sections from preconditioned (P) and tolerant (T) WT brains ( n = 3 each), and preconditioned and
tolerant KO brains (n = 3 and 1, respectively).
Regulation of microRNAs in preconditioned cortex
TA Lusardi et al
753
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 744756
Page 10
transcription and increased mRNA levels, mechan-
isms of preconditioning-induced protein expression
could include posttranscriptional regulation of
mRNAs by miRNAs, consistent with studies showing
miRNA regulation of protein expression indepen-
dent of changes in mRNA levels (Baek et al, 2008;
Selbach et al, 2008).
Functionally, miRNAs expressed within dendrites
regulate translation of proteins mediating dendritic
growth (Schratt et al, 2006). Further, miRNAs are
important for regulating synaptic plasticity, and
miRNAs target (and are targeted by) plasticity
mediators such as CREB, fragile X mental retardation
protein, and MeCP2 (Smalheiser and Lugli, 2009). A
recent study has shown that miR-132 directly
regulates MeCP2 protein expression in rat cortical
neurons: increased miR-132 leads to decreased
MeCP2 protein, whereas decreased miR-132 leads
to increased MeCP2 protein (Klein et al, 2007). This
finding is consistent with our data showing de-
creased miR-132 and increased MeCP2 protein in
ischemic preconditioned mouse cortex. Our micro-
arrays and qRT-PCR both showed miR-132 signifi-
cantly decreased in preconditioned cortex. On the
basis of our target analysis showing that many
miRNAs, including miR-132, are predicted to bind
to the 3
0
UTR of MeCP2 mRNA, we used immunoblot
and immunohistochemistry studies to evaluate
MeCP2 protein expression and show, for the first
time, that MeCP2 is rapidly increa sed in precondi-
tioned co rtex. As miR-132 expression is decreased
and MeCP2 protein increased in preconditioned
cortex, we suggest that MeCP2 mRNA is translation-
ally repressed by miRNAs in control brain and that
preconditioning leads to derepression of MeCP2
mRNA by miRNAs with resultant synthesis of
MeCP2 protein. When we examined the effect of
preconditioning and tolerance in MeCP2 KO mice,
we found increased susceptibility to ischemia,
consistent with studies showing increased cell death
in cerebellar granule neurons in MeCP2 KO mice
exposed to excitotoxicity and hypoxic-ischemia
(Russell et al, 2007). Further, MeCP2 KO mice show
increased susceptibility to hypoxia in telencephalic
neuronal networks that involve disturbed potassium
channel function, suggesting that hypoxia might
contribute to the vulnerability of male Rett patients
who are either not viable or severely disabled
(Fischer et al, 2009). Although these initial studies
in MeCP2 KO mice are not conclusive, they do
provide further evidence that MeCP2 is necessary for
the induction of ischemic preconditioning.
Given that the MCAO treatments are identical except
for duration of occlusion, we expected miRNAs
regulated by preconditioning also to be regulated by
ischemia. We predict that these common miRNAs
mount a response to subsequent injury, but that
cell death pathways induced by the longer duration of
ischemia overcome this response. Although we focused
on miRNAs decreased in preconditioned cortex, many
uniquely regulated miRNAs were increased in precon-
ditioned cortex. These miRNAs could serve to repress
translation of mRNAs not essential for neuroprotection
as a mechanism of energy conservation; studies focused
on the role of these miRNAs in tolerance are currently
in progress. The studies presented herein set the stage
to address additional questions such as which miRNAs
specifically target MeCP2 to regulate protein expres-
sion. Our use of a low-stringency prediction program
(miRanda, version 2005) allowed us to assess the
cooperative potential across miRNA changes. Current
studies examining specific miRNA/MeCP2 interactions
and their therapeutic potential are focused on five
preconditioning-decreased miRNAs predicted by three
increasingly stringent bioinformatic prediction pro-
grams (miRanda, TargetScan, and PicTar) to target
MeCP2 mRNA. In addition, recent studies show that
MeCP2 is not restricted to neuronal cells as previously
thought, but is also expressed in glia (Ballas et al, 2009;
Maezawa et al, 2009). Given that miRNAs can activate
translation in quiescent cells but repress translation in
proliferating cells (Vasudevan et al, 2007), differential
regulation of target proteins could occur in neurons and
glia. W e trust that these studies will contribute to our
understanding of the mechanisms underlying ischemic
preconditioning-induced tolerance, and have potential
to translate into novel strategies for the treatment of
ischemic brain injury: the induction of tolerance.
Acknowledgements
We acknowledge the support of NIH (R21NS054220,
JAS), the NIH Neuroscience Microarray Consortium,
and Dr Holly Dressman, Director of the Duke
University Microarray Facility. We thank Mr Rob
Lusardi (Slowdog Software, Portland, OR, USA) for
creating the miRNA microarray analysis program
used for the data analysis. We thank Jaclyn Shingara
and Dr David Brown of Ambion/Applied Biosystems
for early contributions to miRNA microarrays in rat
brain. RPS envisioned a role for miRNAs in ischemic
tolerance, JAS designed and supervised all experi-
mental aspects of this project, GP and TY performed
mouse neurosu rgeries, JAS isolated all RNAs for
microarray studies and qRT-PCR studies, TAL and
JAS analyzed miRNA microarray data, JQL sectioned
mouse brains, CDF performed immunoblot and
immunohistochemi stry, CLF performed immunoblot,
and maintained the MeCP2 KO mice. JAS, TAL, and
RPS wrote the paper.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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  • Source
    • "The C allele at this locus is associated with increased aggression in schizophrenia , thus implicating the interaction between altered MeCP2 expression and genetic background as a potential mechanism [93]. Increased expression of MeCP2 resulting from reduction of miR-132-mediated repression has also been implicated in the neuroprotective response to pending ischemic injury [94]. These examples highlight how the 3′UTR may serve as a potential source for pathogenic misregulation of MeCP2, and as such, it may be worthwhile to conduct additional studies investigating the potential "
    [Show abstract] [Hide abstract] ABSTRACT: Methyl-CpG-binding protein 2 (MeCP2), encoded by the gene MECP2, is a transcriptional regulator and chromatin-remodeling protein, which is ubiquitously expressed and plays an essential role in the development and maintenance of the central nervous system (CNS). Highly enriched in post-migratory neurons, MeCP2 is needed for neuronal maturation, including dendritic arborization and the development of synapses. Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a debilitating neurodevelopmental disorder characterized by a phase of normal development, followed by the progressive loss of milestones and cognitive disability. While a great deal has been discovered about the structure, function, and regulation of MeCP2 in the time since its discovery as the genetic cause of RTT, including its involvement in a number of RTT-related syndromes that have come to be known as MeCP2-spectrum disorders, much about this multifunctional protein remains enigmatic. One unequivocal fact that has become apparent is the importance of maintaining MeCP2 protein levels within a narrow range, the limits of which may depend upon the cell type and developmental time point. As such, MeCP2 is amenable to complex, multifactorial regulation. Here, we summarize the role of the MECP2 3' untranslated region (UTR) in the regulation of MeCP2 protein levels and how mutations in this region contribute to autism and other non-RTT neuropsychiatric disorders.
    Full-text · Article · Oct 2015
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    • "DNA microarray technology has been an invaluable tool for resolution of the genetic profile of IT. Microarrays were used to study differential gene expression patterns in oxygen–glucose deprived rat hippocampal slices and microRNA expression and regulation of its target MeCP2 in IPC-stimulated mouse cortex [32, 106]. It was also found to be beneficial in an adult mouse model of HPC, where upregulation of cell survival genes like HIF, insulin-like growth factor (IGF), etc., and region-specific expression patterns within the same brain were made apparent [33]. "
    [Show abstract] [Hide abstract] ABSTRACT: Cerebral preconditioning constitutes the brain's adaptation to lethal ischemia when first exposed to mild doses of a subtoxic stressor. The phenomenon of preconditioning has been largely studied in the heart, and data from in vivo and in vitro models from past 2-3 decades have provided sufficient evidence that similar machinery exists in the brain as well. Since preconditioning results in a transient protective phenotype labeled as ischemic tolerance, it can open many doors in the medical warfare against stroke, a debilitating cerebrovascular disorder that kills or cripples thousands of people worldwide every year. Preconditioning can be induced by a variety of stimuli from hypoxia to pharmacological anesthetics, and each, in turn, induces tolerance by activating a multitude of proteins, enzymes, receptors, transcription factors, and other biomolecules eventually leading to genomic reprogramming. The intracellular signaling pathways and molecular cascades behind preconditioning are extensively being investigated, and several first-rate papers have come out in the last few years centered on the topic of cerebral ischemic tolerance. However, translating the experimental knowledge into the clinical scaffold still evades practicality and faces several challenges. Of the various preconditioning strategies, remote ischemic preconditioning and pharmacological preconditioning appears to be more clinically relevant for the management of ischemic stroke. In this review, we discuss current developments in the field of cerebral preconditioning and then examine the potential of various preconditioning agents to confer neuroprotection in the brain.
    Full-text · Article · Jun 2015 · Molecular Neurobiology
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    • " (2C), miR‑448 reduced adipocyte differentiation, again indicating a negative feedback relationship. [20] Reduced adipocyte differentiation may result from miR‑448‑mediated suppression of the transcription factor Krueppel‑like factor 5. [21] MiRNAs also regulate responses to brain ischemia and ischemic tolerance induced by ischemic preconditioning. [22] Transient focal ischemia in adult rat regulated the expression of miRNAs predicted to target proteins involved in signal transduction and ionic homeostasis. [23] A recent study reported (propofol‑regulated) miR‑347 downregulation following ischemic stroke, intracerebral hemorrhage, and kainite‑induced seizures, implicating this analgesi"
    [Show abstract] [Hide abstract] ABSTRACT: Background: Sevoflurane and propofol are widely used anesthetics for surgery. Studies on the mechanisms of general anesthesia have focused on changes in protein expression properties and membrane lipid. MicroRNAs (miRNAs) regulate neural function by altering protein expression. We hypothesize that sevoflurane and propofol affect miRNA expression profiles in the brain, expect to understand the mechanism of anesthetic agents. Methods: Rats were randomly assigned to a 2% sevoflurane group, 600 μg·kg - 1·min - 1 propofol group, and a control group without anesthesia (n = 4, respectively). Treatment group was under anesthesia for 6 h, and all rats breathed spontaneously with continuous monitoring of respiration and blood gases. Changes in rat cortex miRNA expression profiles were analyzed by miRNA microarrays and validated by quantitative real-time polymerase chain reaction (qRT-PCR). Differential expression of miRNA using qRT-PCR among the control, sevoflurane, and propofol groups were compared using one-way analysis of variance (ANOVA). Results: Of 677 preloaded rat miRNAs, the microarray detected the expression of 277 miRNAs in rat cortex (40.9%), of which 9 were regulated by propofol and (or) sevoflurane. Expression levels of three miRNAs (rno-miR-339-3p, rno-miR-448, rno-miR-466b-1FNx01) were significantly increased following sevoflurane and six (rno-miR-339-3p, rno-miR-347, rno-miR-378FNx01, rno-miR-412FNx01, rno-miR-702-3p, and rno-miR-7a-2FNx01) following propofol. Three miRNAs (rno-miR-466b-1FNx01, rno-miR-3584-5p and rno-miR-702-3p) were differentially expressed by the two anesthetic treatment groups. Conclusions: Sevoflurane and propofol anesthesia induced distinct changes in brain miRNA expression patterns, suggesting differential regulation of protein expression. Determining the targets of these differentially expressed miRNAs may help reveal both the common and agent-specific actions of anesthetics on neurological and physiological function.
    Full-text · Article · May 2015 · Chinese medical journal
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