Lasting Epigenetic Influence of Early-Life Adversity on the BDNF Gene

2. Lasting Epigenetic Influence of Early-Life Adversity on the BDNF Gene Tania L. Roth, Farah D. Lubin, Adam J. Funk, J. David Sweatt Presented by Justin P. Smith

3. Background • Early life maltreatment • Changes neural mechanisms • Psych illnesses: MAJOR DEPRESSION, schizophrenia, bi-polar disorder • Stress-induced changes • Neural plasticity in PFC and hippocampus • BDNF protein levels * *not a real baby

4. Background cont. • Epigenetics! • Direct methylation of DNA • Posttranslational modification of histones • Either can + or – gene transcription • Biochem view DNA methylation was a static process (consensus is changing)

5. Animals • Male and Female Long-Evans rats • All behavior testing done during light period* • Food and water ad libitum

6. Design • Maltreatment vs Cross-foster care • 30 min/day for one week (PN1-7) • Gene Assays • Tissue from PFC and Hippocampus collected PN8, PN30 and PN90 • Zebularine– DNA methylation inhibitor • Left lateral ventricle of maltreated adults (7 days) • Zebularine or Vehicle • Maltreated females mated, pups cross-fostered • Controls • PN8, PFC and hippocampus isolated

7. Maltreatment • Limited nesting resources in unfamiliar environment to stress mothers • Pups were: stepped on, dropped, dragged, actively rejected, roughly handled • Littermate controls exposed to + caregiving • 30 min of maltreatment, PN1-7

8. Fig 1 Infants experienced an adverse caregiving environment. (A) Qualitative assessment of the percent occurrence of pup-directed behaviors in the maltreatment condition indicates that pups experienced predominately abusive behaviors, which resulted in considerable audible pup vocalization. (B) In sharp contrast, pups experienced significant amounts of normal maternal care behaviors in the cross-fostered maternal care condition

9. Maltreatment During Infancy Decreases BDNF GeneExpression in the Adult PFC • Assessed BDNF total mRNA levels (exon IX) PFC & hippocampus of adult males and females • Exposed to the maltreatment paradigm as neonates • Suggests hippocampal ↑was not exclusive to the experience of maltreatment, reflective of other variables: • exposure to new caretakers • experience in a novel environment • and/or removal from the biological mother and home cage

10. Fig 2

11. BDNF BDNF gene contains nine 5’ non-coding exons (I-IXA), each linked to a unique promoter that differentially splices to the common 3’ coding exon IX. The activity of each noncoding promoter region dictates differential expression of BDNF exon-specific transcripts, providing tissue-specific and activity dependent regulation of the BDNF gene across development and in adulthood

12. Infancy Maltreatment Elicits a Lasting Increase inBDNF DNA Methylation in the PFC • BDNF gene expression correlated with changes in BDNF DNA methylation • Evaluated exon IV encompassing the transcription start site and (cAMP) response element • epigenetic regulation of this region is gaining support for role in neural activity-dependent BDNF gene expression • Evaluated exon IX, large CpG sites • Exon IV and Exon IX mRNA transcripts ↑ during postnatal development in the cortex and hippocampus • Dynamic methylation of exon IV suggested mechanism mediating BDNF gene expression during development and thus susceptible to environmental insults

13. Fig 3 Maltreated groups only Age:

14. Confirming methylation • Direct bisulfite DNA sequencing PCR (BSP) on site-specific methylation of 12 CpG dinucleotides within the same region of exon IV screened by methylation specific real-time PCR (MSP) • Significant increases in methylation across the region in adults with a history of maltreatment (Fig 4)

15. Fig 4 * *cAMP response element site

16. Deficits in BDNF Gene Expression inthe Adult PFC Rescued by Treatment with a DNAMethylation Inhibitor • Infused zebularine, a DNA methylation inhibitor, left ventricle over 7 days • Sufficient to ↓methylation of BDNF exon IV DNA and rescue both BDNF exon IV mRNA & total mRNA levels in adults with maltreatment history

17. Fig 5

18. Maltreated + Zebularine = no diff from normal control Fig 5

19. BDNF DNA Methylation Patterns in the PFC fromMaltreatment Are Perpetuated to the Next Generation

20. Fig 6 Females with a history of maltreatment display aberrant maternal behavior toward their own offspring “There’s your problem”

21. Fig 7 • A) Cross-fostering mal-offspring to a female with a history of normal infancy (Mal-Normal)↑ site 1 • B) Reverse methyl @ site 2 • C) Cross-fostering ↓ methyl but still ↑ over normal

22. Fig 8 • Prepartum behavioral observations • indicate that females with history of maltreatment displayed • significantly more anxiety-related behaviors

23. Conclusion • Infant maltreatment results in methylation of BDNF DNA through the lifespan to adulthood, reduced BDNF gene expression in the adult PFC • Altered epigenetic marks and gene expression in the adult can be rescued with chronic treatment of a DNA methylation inhibitor (zebularine) • Abused rats grow up and mistreat their own offspring and their offspring also have significant DNA methylation • Inability of cross-fostering to completely rescue CNS DNA methylation

24. Thank you