Parental methamphetamine exposure affects offspring’s behavior and DNA methylation Juan I. Young John P. Hussman Institute for Human Genomics Dr. John T. Macdonald Foundation Department of Human Genetics *SCOR*NIH/NIMH No conflict of interest to declare

Heritable transmission of environmentally induced phenotypes: multigenerational inheritance Studies suggest the transmission of behavioral traits acquired by one generation to subsequent generations through epigenetic processes Phenotype APhenotype B Exposure to a drug of abuse (methamphetamine) Phenotype B Multigenerational persistence

Methamphetamine is a major drug of abuse in many parts of the world. Current use of METH surpasses the use of cocaine and opiates. Methamphetamine addiction is associated with psychotic behavior and long term cognitive impairment. Surveys of past-month illicit drug use among females aged showed that 6-7% of these women were pregnant, and continued to use drugs during all three trimesters of pregnancy; 2,000-3,000 abused Methamphetamine. Environmetal intervention: Exposure to metamphetamine

A major concern is the prospect that parental methamphetamine abuse and in utero exposure may have debilitating effects on generations that are either indirectly exposed, or even unexposed to the drug. Exposure: simulated the human pattern of parental METH exposure. –Male and female mice (F0) were exposed to an intermittent escalating regiment of METH or saline from adolescence through adulthood and during pregnancy. Environmetal intervention: Exposure to metamphetamine

F1 phenotype: motivational effects of cocaine Molecular Psychiatry, (18 February 2014) Long lasting effect of methamphetamine on CPP (sexually dismorphic) Male pups insensitive to maternal influence Female pups sensitive to maternal effects In utero: S S S S M M M M Fostering dam: S M S M S M S M

Molecular Psychiatry, (18 February 2014) In utero: S S S S M M M M Fostering dam: S M S M S M S M F1 phenotype: associative learning and memory Long lasting effect of methamphetamine on fear conditioining Sensitive to maternal effects : Fostering of METH pups by saline dams restored dampened freezing of METH pups

Molecular Psychiatry, (18 February 2014) F1 phenotype: activity and anxiety Long lasting effect of methamphetamine-induced maternal effect Maternal care of METH dams had a significant sex- dependent effect on anxiety-like behavior of saline offspring In utero: S S S S M M M M Fostering dam:S M S M S M S M In utero: S M S M Fostering dam:S M M S

F1 summary (Behavior) Prenatal METH exposure produces long-lasting changes in the offspring’s behavioral phenotypes. Effects are phenotype dependent:  CPP: Sexual dimorphism  Maternal influence on the phenotype (independent of dam treatment) also sexually dimorphic  Fear conditioning: Maternal influence on the phenotype dependent on dam treatment (“saline rescue”)  Open field: Maternally influenced phenotye (dependent on dam treatment (“METH induced”)  Light/dark box:Maternally influenced phenotye (dependent on dam treatment (“METH induced”) and sexually dimorphic

Mechanism by which prenatal exposures affect long term phenotypes One possible mechanism involves alterations of DNA methylation marks in the genome. compared the DNA methylome of the hippocampus of the F1 SpSd, MpMd, SpMd and MpSd mice by MeDIP- microarray NimbleGen microarray: covers 15,936 UCSC annotated CpG islands and all RefSeq gene promoter regions

The group that exhibited the highest number of identified peaks was MpMd, suggesting that the combination of in utero exposure to METH and METH-induced maternal care mostly promotes DNA methylation. A comparison of MpMd vs. SpSd samples identified 1822 methylation peaks aligning to gene promoter regions as differentially methylated regions (DMR). The majority of these DMR showed hypermethylation in MpMd, as compared to SpSd. F1 epigenotype: DNA methylation in hippocampus

Validation: 75% of selected DMR showed methylation differences by bisulfite sequencing concordant with the array data. More variability in methylation levels in samples from maternally METH exposed mice than in SpSd samples, suggesting that the response of hippocampal cells to maternal METH exposure is heterogeneous. F1 epigenotype: DNA methylation in hippocampus Molecular Psychiatry, (18 February 2014)

EMR: No effect of paternal METH on pup DMR: effect of METH on maternal care EMR: No effect of METH on maternal care SpSd vs MpSd DMR: effect of paternal METH on pup under the care of a saline dam SpMd vs MpMd DMR: effect of paternal METH on pup under the care of a METH dam SpSd vs MpMd DMR: combination of effect of paternal METH on embryo and effect of METH on maternal care SpSd vs SpMd DMR: effect of METH on maternal care (on saline embryos) MpMd vs MpSd DMR: effect of METH on maternal care on METH embryos SpSd vs MpMd DMR: combination of effect of paternal METH on embryo and effect of METH on maternal care MpMd vs MpSd SpMd vs MpMd DMR: effect of paternal METH on pup Paternal METH effects on the F1 pup’s methylome independent of maternal care influences. Paternal METH effects on the F1 pup’s methylome mediated by altered maternal care : : : : Numbers in black: hypermethylated DMR; numbers in red: hypomethylated DMR; EMR: equally methylated region.

Methylation induced by METH- exposure (insensitive to maternal modulation) 62 Adora1 (M & C) Ilk (C) Akap5 (C) Demethylation induced by METH- exposure (insensitive to maternal modulation) 35 Camkk2 (M) Methylation induced by METH-altered maternal care 70 Pcdh8 (C) Pomc (H) Cln8 Rtn1 Tbx5 Demethylation induced by METH-altered maternal care 39 Camk2a (M&C) Methylation induced by interaction of METH- exposure and METH-altered maternal care 60 Gsk3b (C) Demethylation induced by interaction of METH- exposure and METH-altered maternal care 22 Hdac5 (M&C) Prenatal METH exposure induce changes in several genes shown to be modulated by: direct exposure to drugs of abuse, and maternal effects. F1 epigenotype: DNA methylation in hippocampus

Top 10 enriched histone terms for the hypermethylated DMR METH-induced methylation, which should produce gene silencing, occurs in promoters that have a histone modification signature of active transcription. NameP-valueZ-score Score 1 H3K4me3 H13.93e H3K4me3 H1 Derived Mesenchymal Stem Cells 1.10e H3K4me3 H9 1.68e H3K4me3 iPS DF e H3K4me3 CD8 Naive Primary Cells 1.59e H3K4me3 iPS DF e H3K4me3 CD19 Primary Cells 7.63e H3K9ac H1 4.42e H3K4me3 H1 BMP4 Derived Trophoblast Cultured Cells 7.84e H3K4me3 IMR901.77e

METH-induced demethylation was observed in promoters associated with an epigenetic signature of transcriptional silencing. NameP-valueZ-scoreScore 1 H3K27me3 Fetal Lung 8.73e H3K27me3 H1 BMP4 Derived Trophoblast Cultured Cells 3.51e H3K27me3 H H3K27me3 Colonic Mucosa H3K27me3 Stomach Smooth Muscle H3K27me3 CD4 Memory Primary Cells H3K27me3 CD4 Naive Primary Cells H3K27me3 CD3 Primary Cells H3K27me3 Penis Foreskin Keratinocyte Primary Cells H3K27me3 CD8 Memory Primary Cells Top 10 enriched histone terms for the demethylated DMR.

F1 summary Prenatal METH exposure produces long-lasting changes in the offspring’s brain epigenome that could contribute to the initiation and maintenance of the observed behavioral phenotypes. Our experiments indicate a significant influence of maternal effects on the epigenotype and behavioral phenotype of the F1 progeny. Parental METH exposure has important consequences on the hippocampal transcriptional landscape of the progeny; promoters that are usually active become methylated while inactive promoters are demethylated.

Aknowledgements Yossef Itzhak Ian Ergui Michael kaplan Vladimir Camarena