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)

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

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)

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

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

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

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

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

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 de-methylation 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