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 Stress-induced changes
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 Gene Assays
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. Exposed to the maltreatment paradigm as neonates
Maltreatment During Infancy Decreases BDNF Gene Expression 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. BDNF gene expression correlated with changes in BDNF DNA methylation
Infancy Maltreatment Elicits a Lasting Increase in BDNF 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 in the Adult PFC Rescued by Treatment with a DNA Methylation 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 from Maltreatment 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. B) Reverse methyl @ site 2
Fig 7
A) Cross-fostering mal-offspring to a female with a history of normal infancy (Mal-Normal)↑ site 1
B) Reverse 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