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

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

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)

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

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

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

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

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

Fig 2

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

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

Fig 3 Maltreated groups only Age:

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)

Fig 4 * *cAMP response element site

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

Fig 5

Maltreated + Zebularine = no diff from normal control Fig 5

BDNF DNA Methylation Patterns in the PFC from Maltreatment Are Perpetuated to the Next Generation

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

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 methyl @ site 2 C) Cross-fostering ↓ methyl but still ↑ over normal

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

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

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