Download Document

P11
Antidepressant treatment is associated with
epigenetic alterations in the promoter of P11 in a
genetic model of depression
Philippe A. Melas, Maria Rogdaki, Andreas Lennartsson, Karl Bjo¨rk,
Hongshi Qi, Anna Witasp, Martin Werme, Gregers Wegener, Aleksander A. Mathe´,
Per Svenningsson and Catharina Lavebratt
Presented by: Justin P. Smith
P11 Background
• Established function of intracellular trafficking
of transmembrane proteins to cell surface
• linked with depression in both humans and
animal models
• ↓ levels in post-mortem brain tissues of
depressed individuals
– P11 knockout (KO) mice display a depression-like
phenotype
P11 continued
• MAOIs & ECT (antidepressants) ↑ levels in
rodent PFC
• SSRIs effect the gene, P11 abundantly
expressed in hippocampal GABAergic
interneurons that also expressed 5-HT1B&4
• P11 interacts with serotonin receptors
• P11 gene therapy in mice NAc effective in
reversing depressed behaviors
P11 relation to 5-HT1B receptors
Methylation Models
• Single CpG methylation model
– distinct DNA elements [usually transcription factor
(TF)-binding sites] whose methylation or
demethylation usually leads to gene silencing or
activation
• Bulk CpG methylation model
– numerous CpG sites and regards high mean
methylation levels as a determinant of inactive
chromatin structure that negatively modifies gene
expression
Methylation Enzymes
• 3 main enzymes: DNMT1, DNMT3a and
DNMT3b (b only during embryonic development)
• DNA methylation in adult mice forebrain
neurons maintained through the action of
DNMT1 and DNMT3a
– (↓ gene expression)
•
•
•
•
LSD1 coordinats histone methylation and DNA
methylation
Methylated Dnmt1 is metabolically unstable
LSD1, by acting directly on both histone H3 and Dnmt1,
causes H3K4 demethylation & ↑ Dnmt1 & DNA
methylation,
Results in chromatin condensation & gene silencing
• H3K4 demethylation by KDM1B creates docking sites for Dnmt3L
• Recruits &/or activates Dnmt3a
• Dnmt3a puts methyl groups on DNA at imprinted loci
Study Aims
1. Levels of P11 in PFC of a genetic rodent
model of depression
2. Is P11 regulation influenced by epigenetic
modifications
3. P11 expression and DNA methylation changes
in response to chronic administration of
escitalopram (Lexapro)
Animals:
Flinders Rats
• Flinders Sensitive Line (FSL)
– Selectively bred putative animal model of depression
– Exhibits ↓ appetite and psychomotor function but exhibits
normal hedonic responses and cognitive function
– sleep and immune abnormalities observed in depressed
individuals
– Neurochemical and/or pharmacological evidence suggests FSL
rat exhibits changes consistent with the cholinergic,
serotonergic, dopaminergic, NPY, and circadian rhythm models
but not the noradrenergic, HPA axis or GABAergic models of
depression
• Control: Flinders Resistant Line (FRL) exhibits changes
consistent with the serotonergic hypothesis of depression
Methods
•
•
•
•
•
•
PFC dissected and frozen -70C
Gene expression in-situ hybridization
Western Blotting
qRT-PCR
Site-specific DNA methylation quantification
Whole-genome methylation analysis
Results
• P11 difference between FSL & FRL
• FSL ↓ P11 mRNA
• FSL ↓ P11 protein
Fig. 1
In-situ mPFC
mRNA
Protein
Fig 2
Putative androgen receptor (AR) binding site
P11 transcription start site
gap region, bases 498–877,
Ar mRNA levels do not
underlie the depressed
phenotype
Fig 3
corresponding to the putative AR binding site
• P11 mRNA levels were significantly
increased in the escitalopramtreated groups
• mRNA level of the FSL-Esc group
was not statistically different
from FRL
• FSL-Esc group was now
statistically similar to FRL
Fig 3
Bulk methylation model
Gap region (Fig 2b) + AR
binding site
• group differences same as
putative AR binding site
Whole-genome methylation analyses
• No global methylation
level was associated
with escitalopram
treatment
Fig 4
Candidate enzymes associated with the observed DNA methylation changes
• Dnmt1 and Dnmt3a levels were
significantly decreased following
treatment with escitalopram in FSL
Fig 4
genes encoding proteins suggested to be involved in DNA demethylation
p=0.06
• In line with
GADD45b’s
demethylating
role in the
adult brain
Conclusions
• Decreased P11 in the PFC of a genetic model
of depression
• ↓ mRNA and protein levels of P11 in the PFC
of FSL compared to FRL
• P11 mRNA between naive FSL & FRL not
statistically significant with qRT-PCR
– ↑ cellular resolution with in-situ hybridization
Conclusions cont.
FSL are hypermethylated in the P11 promoter region
• AR most relevant TF binding site
• AR androgen responsive TF inhibits CRF &
leads to ↓ HPA activity
• Sex differences in depression, sex hormones
involved in disease(?)
• No Ar mRNA diffs in this study BUT
transcriptionally down-regulated in
postmortem brains of depressed individuals
– Possible post-translational modifications
P11 promoter & functional
experiments needed
• detected ↑ methylation levels at putative
binding site of AR in FSL (Single critical model)
• Same when tested 5 CpG sites (Bulk model,
Fig 3c)
• P11 activity probably regulated by other TFs
and co-activators, not just AR
Escitalopram & methylation decrease
• Esc treatment reversed FSL methylation to FRL
pattern
• SSRI ↓ in P11 methylation not accompanied
by genome-wide hypomethylation
• ECT gives site-specific, NOT global demethylation in brain
– What gives ECT this specificity?
Dnmt1 and Dnmt3a levels
• Dnmt1 and Dnmt3a –methyltransferases that
maintain methylation activity
• Dnmt1 and Dnmt3a both down-regulated in
FSL-Esc group
• ↓ methylation ↑ gene expression
GADD45b -a demethylase candidate
• trend for↓ Gadd45b levels in FSL animals
• MBD2, MBD4 & AID no significance
• Gadd45b induced by ECT & promotes DNA
demethylation in adult brain
• GADD45b in FSL possible answer for
unexplained basal hypermethylated pattern
observed between untreated groups
– FSL vs FRL Fig. 3b, c
Take home
• P11 role in depression
• Epigenetic control of P11
• ↓ P11 in FSL’s ↑ methylation at promoter
region
• Esc rescued FSL
– ↑ P11, ↓ methylation plus ↓ Dnmt1 and Dnmt3a
Thank you