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P11

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´,

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P11

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  1. P11

  2. Antidepressant treatment is associated withepigenetic alterations in the promoter of P11 in agenetic 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

  3. 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

  4. 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

  5. P11 relation to 5-HT1B receptors

  6. 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

  7. 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)

  8. 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

  9. Study Aims • Levels of P11 in PFC of a genetic rodent model of depression 2. Is P11 regulation influenced by epigenetic modifications 3. P11expression and DNA methylation changes in response to chronic administration of escitalopram (Lexapro)

  10. 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

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

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

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

  14. 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

  15. Fig 3 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

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

  17. 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

  18. 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

  19. Conclusions cont.

  20. 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

  21. 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

  22. 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?

  23. 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

  24. 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

  25. 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

  26. Thank you

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