Epigentic mechanism

1. Insights into the epigenetic mechanisms involving
histone lysine methylation and demethylation in
ischemia induced damage and repair has therapeutic
implication
AUTHOR
Sumana Chakravarty , priya Jhelum , Unis ahemad bhat , Wenson D Rajan Swati
maitra ,Salil S Pathak , Anant B Patel , Arvind Kumar
Published in –ELSEVIER
PRESENTED BY
ASHUTOSH B. MAHALE
M.PHARMACY FIRST YEAR ( PHARMACOLOGY )
UNIVERSITY DEPARTMENT OF PHARMACEUTICAL SCIENCES, R.T.M UNIVERSITY,NAGPUR

2. INTRODUCTION
• stroke is caused due to interruption of cerebral blood supply
,and 13-25%stroke are internal carotid artery occlusion
• An establishment of Internal carotid artery occlusion (ICAO)
model in CD1 mouse that result in mild to moderate level of
neuronal damage most striatum
• Epigenetic mechanisms in stroke control the transcription
events as DNA methylation and histone lysine acetylation
• The epigenetic mechanisms like H3 and H4 methylation and
demethylation in H3K9, H3K27 and H4K20 methylation , a
transcriptionally repressive epigentic modification implicated
in stroke
• Histone lysine methyltransferases (KMT) and histone lysine
demethylases (KDM) have promising role in ischemia induced
stroke

3. • The study involved ICAO model and novel role of
epigenetic regulatory mechanisms in H3K9me2 in
striatal damage and subsequent repair after mild to
moderate ischemia
• A novel insight was use of an inhibitor of jmjd2
class lysine demethylase to prevent H3K9me2 level
go down after induction of ICAO result in reduction
of neurological ICAO giving neuroprotection

4. OBJECTIVE
• To established a mild to moderate stroke model
(ICAO) in CD1 mouse to Investigate the role of
histone lysine methylation and demethylation in
ischemia induced brain damage and subsequent
recovery at various time points

5. MATERIALS AND METHODS
1. Animals
2. Internal carotid Artery Occlusion
3. Neurological deficit score –
0-normal
1-inflammation of eyelid/ptosis
1.5- both
2 -less body bending
2.5-consisting body bending
3-1+2both
4-3+less griping
5-3+weak circling towards the paretic side
4. Forelimb grip strength test
5. Rotarod performance test
6. Histological staining –two types
TUNEL- apoptic cell death – stain fragmented nuclear DNA in dead cell
Hematoxylin and eosin (H#E) –to visualize vacuolization

6. 6. Brain tissue fixation and sectioning
7.Histological Staining
8. Measurement of apoptotic cell index –
9. Magnetic resonance imaging (MRI)-
10. Reverse transcriptase-quantitative polymerase chain
reaction (qt-PCR)
11. Western blotting-
12. Chromatin immunoprecipitation (chip) assay
13. Dimethyloxaloylglycine (DMOG) administration to
mice after ICAO
14.Digoxin (DIG) administration
15. Statistical analysis

7. RESULTS
1. Magnetic resonance imaging (MRI) showed an
observable neural damage in striatum at 1 day post
–ICAO
(A)Representative MRI
mages(MRI)shows prominent ischemic lesion at ipsilateral side of the striatum as compared with contralateral side
(non-ischemic region)

8. 2. TUNEL hematoxylin and eosin staining revealed cellular death or
damage mostly to striatum
1H MRS spectra from striatum showing reduction in NAA, choline,
creatine, taurine and increase in lactate level on the ipsilateral side as
compared with that in contralateral side of lesion

9. TUNEL staining showing apoptotic cell death labeled by green fluorescence TUNEL positive
cell at 1 d post-ICAO in affected (ipsilateral side) and nuclei stained with propidium iodide
(red fluorescence). However, TUNEL positive cell were not observed in sham group . The
apoptotic index, determined by the ratio of TUNEL positive cells and total cells, expressed in
percentage was increased in ICAO group
(C) In H&E staining, the upper panel shows H&E stained tissue section. Enormous
vacuoles are visible in ICAO group. Lower panel shows statistical representation of normal
vs. vacuolar area, calculated from H&E stained sections from both sham and ICAO group
(n = 5, *p b 0.05 compared to sham)

10. 3. Behavioral functional outcomes post ICAO
assessed by neurobehavioral deficit score (NDS) , grip
strength and rotarod test

11. (A) Neurological deficit score (NDS) were performed at 6 h, 1 d, 3 d,7 d and 15 d post-ICAO, n = 7 in
each group(*p b 0.05, **p b 0.01 compared to 6 h and #p b 0.05, ##p b 0.01 compared to 1 d (B) Grip
strength test performed at 1 d, 3 d and 7 d post-ICAO, n = 7,**p b 0.01 compared to sham,##p b
0.01compared to 1 d (C)Rotarod performance test performed at 1 d,3 d and7 d post-ICAO n = 7,**p
b 0.01compared to sham ,#p b 0.05 compared to1 d.

12. 4.ICAO induced striatal damage was associated with increase in
hypoxia inducible factor 1ᾳ and inflammatory cytokines
The role of Hypoxia-inducing factor(HIF-1α)and inflammatory gene sin ICAO
model.(A and B)Hif-1α an mRNA expression increases at early time period 1d of
post-ICAO and few inflammatory marker sp65,TNF-α,IL-1α and IL-6mRNA
expression increases at 1d of post-ICAO as compared to the sham ipsilateral group

13. blot images and densitometry quantification shows increased expression ofHIF-1α at 3 h of
post-ICAO and return to basal level at 1 d of post-ICAO as compared to the sham ipsilateral
group. β-actin were using as a loading control. Protein bands were quantified by
using ImageJ analysis and normalized by β-actin. Statistical analyzers were carried out
by using Student’s t-test, n = 6 to 8 mice ineachgroup.*p b 0.05,and **p b
0.01versusSham group
5.ICAO induced neural damage and subsequent recovery was associated with
transcriptional dysregulation of epigenetic regulators histone lysine
methyltranserases (KMT)

14. Gene expression levels study of various histones lysine methyltransferase(KMTs) genes Gene
expression study shows dysregulation of epigenetic regulators at various time periods after
ICAO as compared to the sham ipsilateral group. Statistical analysis was carried out by using
Student's t-test, n = 6 to 8 mice in each group. *p b 0.05 and **p b 0.01 versus Sham
ipsilateral group

15. . Representative Immunoblot images of H3K9me2, H3K27me2, and H4K20me3 at different
time periods of post-ICAO and β-actin were used as a loading control (upper panel). Protein
bands were quantified by using ImageJ analysis and normalized by β-actin (lower panel).
Statistical analysis was carried out using Student's t-test. *p b 0.05 (n = 5 to 6 in each
group)compared to sham ipsilateral group

16. 6.ICAO induced change in striatum was also associated with
dysregulation in transcription of few histone lysine demethylases
Gene expression level study of various s histone lysin demethylase(KDMs) Gene expression
study shows dysregulation of epigenetic regulators at various time periods after ICAO as
compared to the sham ipsilateral group. Statistical analysis were carried out by using
Student's t-test ,n = 6 to 8 mice in each group. *p b 0.05and **p b 0.01 versus Sham group

17. 8.Transcriptional activation of neuroinflammatory genes IL-1ᾳ,TNF-ᾳ
and p65 in striatum was due to attenuation of H3K9me2 level on their
promoters
7. Chromatin immunoprecipitation (ChIP) assay on pooled ipsilateral striatal
tissue (n = 3) at 1 d post-ICAO. ChIP –qPCR data shows a decrease in
H3K9me2 level at inflammatory gene promoters' p65, IL-1α and TNF-α in
ICAO group as compared to its sham group

18. 9.Preventing the attenuation of H3K9me2in striatum right after by
blocking the activity of Jmjd2 class demethylases had neuroprotective
effects
(A)DMOG treatment reduces
neurological severity score at 1 d
post-ICAO
(B)Representative image of TUNEL staining showing
ischemia induced cell death as detected by green
fluorescence stained TUNEL positive cells and nuclei
stained with propidium iodide (red fluorescence).
Quantitative analysis showed that, apoptotic index
percentage determined by the ration of TUNEL positive
cells and total cells were significantly less in DMOG
treated ICAO group as compared to vehicle treated ICAO
group

19. (C) At mRNA level, DMOG stabilizes
HIF-1α expression and reduces
Casp3 and Bax expression as
compared to vehicle grou (C) Representative western blot and
densitometryquantification of protein bands shows an
increase in H3K9me2 protein expression inICAO + DMOG
and ICAO+ DIG + DMOG groups as compared to ICAO +
Vehicle group. Statistical analysiswascarried
outbystudent's t-test; n = 5 *p b 0.05 ascompared to
vehicle group.

20. Discussion
• ICAO involve a milder neurological deficits that may
asymatomatic to sympatomatic in stroke
• The MRS analysis after ICAO revealed a significant increase in
level of lactate and reduction in level of NAA, creatine , choline
indicate ischemia induced damage and neuronal death
• Cell death assay by tunnel staining show more fragmented DNA
on ipsilateral side of striatum compared to contralateral side at
1day ICAO
• H and E staining show more vacuolization on ipsilateral side
• Based on forelimb and rotarod seems that ICAO induced motor
impairment coordination at 24hr and persists till 3d post ICAO
• ICAO induced HIF-1ᾳ expression at 3 hr but get down at 1 day
post ICAO compared to sham group
• Gene expression of p65,IL, TNF-ᾳ was up in 1 day post ICAO show
role in cellular damage in stroke

21. • Investigation shows role of KMTs and jumonji domain
containing KDMs in regulation of epigenetic modification
H3K9me2 in neural damage and subsequent repair in
striatal of CD1mouse
• The H3K9me2 a transcriptionally repressive epigenetic
mark, was down at 1day post ICAO and recovered at 15days
post ICAO correlated with recovery in stroke
• Reduction on level of jmjd1 and jmjd2 at 3day post ICAO
help in methylation at H3K9 and recovery in neuronal
damage
• The DMOG , inhibitors of histone lysine demethylases jmjds
induced increase in H3K9me2 level after ischemic attack
reduced damage and initiating neuronal regeneration