2. P11

3. 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

4. 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

5. 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

6. P11 relation to 5-HT1B receptors

7. 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

8. 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)

9. H3K4 demethylation by KDM1B creates docking sites for Dnmt3L
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

10. Study Aims
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)

11. 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

12. Methods PFC dissected and frozen -70C
Gene expression in-situ hybridization
Western Blotting
qRT-PCR
Site-specific DNA methylation quantification
Whole-genome methylation analysis

13. Results P11 difference between FSL & FRL FSL ↓ P11 mRNA
FSL ↓ P11 protein
Fig. 1
In-situ mPFC
mRNA
Protein

14. Fig 2 Ar mRNA levels do not underlie the depressed phenotype
Putative androgen receptor (AR) binding site
P11 transcription start site
gap region, bases 498–877,

15. Fig 3
corresponding to the putative AR binding site
P11 mRNA levels were significantly increased in the escitalopram-treated groups
FSL-Esc group was now statistically similar to FRL
mRNA level of the FSL-Esc group was not statistically different from FRL

16. Fig 3 group differences same as putative AR binding site
Bulk methylation model
Gap region (Fig 2b) + AR
binding site
Whole-genome methylation analyses
group differences same as putative AR binding site
No global methylation level was associated with escitalopram treatment

17. Fig 4
Candidate enzymes associated with the observed DNA methylation changes
Dnmt1 and Dnmt3a levels were significantly decreased following treatment with escitalopram in FSL

18. In line with GADD45b’s demethylating role in the adult brain
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

19. 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

20. Conclusions cont.

21. 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

22. 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

23. 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 de-methylation in brain
What gives ECT this specificity?

24. 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

25. 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

26. 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

27. Thank you