1. Epigenetic Status of GDNF in the Ventral Striatum Determines Susceptibility and Adaptation to Daily Stressful Events

2. Study Aim
“…clarify the molecular mechanisms underlying the susceptibility and adaptation to chronic stress using stress-vulnerable BALB and stress-resilient B6 mice strains.”

3. Study Organisms
Inbred BALB/c (BALB) mice – bred to be really high strung.
stress-vulnerable
C57BL/6 (B6) mice – bred to be really laid back
Stress-resistant

4. How do you measure stress in mice?
Forced Swim Test
Measure time to as well as length of immobility
Sucrose Preference Test
Measure preference for sucrose water or water
Social Interaction Test
Measure amount of social interaction
Novelty-suppressed Feeding Test
Measure amount of time it takes to eat in a novel environment
Elevated Zero Maze Test
Measure time spent in open sections.

5. Can you tell which one is a maze?
Apparently neither can neuroscientists.

6. Chronic Ultra-Mild Stress
Sequential exposure to various mild stressors
Nociceptive stressors and food and water deprivation are not included in regimen.
Includes environmental and social stressors.

7. CUMS procedures 3 stressor categories
Diurnal (2 of 5 applied randomly) – cage tilts, small cage, paired housing, soiled cage, odor
Nocturnal – difficult food access, cage tilt, permanent light, soiled caged
Reversed light dark schedule for a few days

8. Chronic Ultra-Mild Stress
Behavioral Response to CUMS
BALB mice showed significant stress responses, except in the elevated zero maze, to chronic stress.
B6 mice showed no significant responses to chronic stress.

9. Corticosterone levels
Also looked at Corticosterone levels as an indicator of stress
Increased Corticosterone (CORT) levels in both BALB and B6 mice 60min after stressor on day 3.
Reduction in CORT levels 60min after a stressor on day 38 in B6 mice. No such reduction in BALB mice

10. Results
BALB mice responded to CUMS with an increase in “depression-like phenotypes”
Sign of a strain susceptible to stress
B6 mice responded to CUMS with a decrease in “anxiety-related behaviors”
Sign of a strain that is adaptive to stress

11. Expression Analyses of a Variety of Neurotrophic Factors in a Mouse Model of Depression
Record mRNA levels in various brain regions in stressed BALB mice
Bdnf – brain derived neurotrophic factor
Gdnf – Glial cell line derived neurotrophic factor
Vegf – vascular endothelial growth factor
Nt-3 – Neurotrophin-3
Nt-4/5 – Neurotrophin-4/5
Cdnf – Conserved dopamine neurotrophic factor
Ngf – Nerve growth factor
Fgf2 – fibroblast growth factor-2
Igf1 – insulin growth factor-1

12. Neurotrophic factors in various stress related brain regions in Stressed BALB mice

13. Neurotrophic Factors cont.

14. Role of GDNF in the Nucleus Accumbens (NAc)
Goal
Investigate if a correlation exists between GDNF expression in the vSTR and depression-like behavior

15. Role of GDNF in the Nucleus Accumbens (NAc)
Goal
Investigate the role of GDNF in depression like behaviors.
Procedure
Injected GDNF gene complex into NAc to overexpress GDNF

16. Role of GDNF in the Nucleus Accumbens (NAc) in depression-like behaviors
Findings
Non-stressed B6 mice showed an increase in social interaction but no change in sucrose preference
Stressed BALB mice showed increased social interaction and sucrose preference.
Possible sign that transcriptional regulation of GDNF in NAc plays a role in susceptibility and adaptation to CUMS

17. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment
Resequence analysis of GDNF promoter showed no difference b/n BALB and B6 mice, suggesting epigenetic regulation for differing expression b/n strains
ChIP analysis revealed several differences in histone modification in both BALB and B6 mice following CUMS and/or cont. IMI treatment.

18. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
BALB mice
significantly less GDNF promoter-containing DNA fragments in acetylated histone 3 (H3ac). This effect was reversed with IMI.
Acetylated histone 4 (H4ac) levels were not affected by CUMS or cont. IMI treatment.

19. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
BALB mice cont.
Also examined CUMS effect on levels of trimethylated histone 3 at lysine 27 (H3K27me3) and trimethylated histone 3 at lysine 4 (H3K4me3)
H3K27me3 not affected by CUMS or IMI
H3K4me3 was significantly reduced by CUMS, but reduction was reversed with IMI

20. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
Stressed B6 mice
H3ac levels at GDNF promoter were significantly increased
H4ac levels showed no significant change

21. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
B6 mice
H3K27m3 levels were significantly reduced by CUMS
H3K4m3 levels were significantly reduced by CUMS

22. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
Measured mRNA levels of Histone deacetylases (HDAC)s 1-11 in BALB mice.
HDAC2 was significantly increased in mice, effect was reversed with IMI

23. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
No significant change in HDAC2 levels in BALB hippocampus or the ventral striatum (vSTR) in B6 mice.
Results suggest that HDAC2 may be a regulator of epigenetic repression of GDNF expression in the vSTR of stressed BALB mic

24. Regulation of Histone modification by CUMS and cont
Regulation of Histone modification by CUMS and cont. Imipramine treatment cont.
Does CUMS influence binding of HDAC2 to GDNF promoter?
BALB mice
GDNF promoter-containing DNA fragments are enriched in HDAC2 immunoprecipitates, effect was reversed with IMI.
No change noted in the BDNF promoter II region
B6 mice
No significant CUMS effect on HDAC2 binding to GDNF promoter.

25. Where we are so far…
Data to this point shows that CUMS increases HDAC2 expression in the vSTR of BALB mice but not in B6 mice.
Hypothesis is that the effect may be important for the transcriptional repression of GDNF and the behavioral susceptibility to CUMS
Tested by a systemic administration of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor.
SAHA tested for antidepressent effects alongside IMI and FLX, both SSRIs

26. Rapid Antidepressant Effects of SAHA on CUMS induced behavioral deficits.
Results
SAHA, but not IMI or FLX mice, showed increased social interaction times and sucrose preference in comparison to vehicle treated controls.

27. Rapid Antidepressant Effects of SAHA on CUMS induced behavioral deficits.
SAHA reduced latency to feeding in novelty-suppressed feeding test in both stressed and non-stressed mice. IMI and FLX had no such effects.

28. Rapid Antidepressant Effects of SAHA on CUMS induced behavioral deficits.
SAHA significantly reduced immobility time in FST, IMI and FLX had no such effect compared to stressed and non-stressed vehicle control mice

29. Rapid Antidepressant Effects of SAHA on CUMS induced behavioral deficits.
SAHA increased mRNA levels of GDNF in the vSTR of stressed mice, IMI and FLX had no such effect.

30. Role of HDAC2 in Behavioral Responces
Goal
Test the direct contribution of HDAC2 in the Nucleus Accumbens (Nac) to CUMS induced depression like behavior.
Accomplished with magic…or some complex target gene. transfers involving viruses

31. Role of HDAC2 in Behavioral Responses
Procedure 1
NAc in BALB mice was bilaterally injected with either AAV-dnHDAC2 (under-express) or AAV-EGFP (control)
Results
AAV-dnHDAC2 Increased social interaction times and increased sucrose preference. Also significantly increased GDNF mRNA levels in the NAc
Strongly suggests that “CUMS induced activation of HDAC2 represses GDNF transcription in the NAc which leads to aberrant depressive behavior in BALB mice”

32. Role of HDAC2 in Behavioral Responses

33. Role of HDAC2 in Behavioral Responses
Procedure 2
NAc of B6 mice were bilaterally infected with either AAV-HDAC2 (over express) or AAV-EGFP (control)
Results
AAV-HDAC2 mice didnt show a significant reduction in interaction time or sucrose preference in comparison to AAV-EGFP mice. Neither was there a difference in GDNF expression.

34. Role of HDAC2 in Behavioral Responses

35. Role of HDAC2 in Behavioral Responses
Procedure 3
NAc of stressed B6 mice were bilaterally infected with either AAV-HDAC2 C262/274A (mutant form, repressed transcription) or AAV-EGFP (control)
Results
Reduction in social interaction time and reduced GDNF expression

36. Role of HDAC2 in Behavioral Responses

37. Role of HDAC2 in Behavioral Responses
Procedure 4
NAc of nonstressed B6 mice bilaterally injected with AAV-HDAC2 C262/274a or AAV-EGFP
Results
No change in social interaction time or GDNF expression compared to control
Procedure 5 and 6
NAc of nonstressed BALB mice injected with AAV-HDAC2, AAV-HDAC2 C262/274a, or AAV-EGFP
No change in social interaction time, sucrose preference, or GDNF levels

38. Role of HDAC2 in Behavioral Responses
Findings
CUMS-induced activation of HDAC2 represses GDNF transcription in the NAc, which results in aberrant behavioral responses in BALB mice
Gain of function of HDAC2 in B6 mice leads to a lack of active response to CUMS.
Other molecular mechanisms modulated by CUMS may also be involved in the HDAC2-mediated Gdnf repression and subsequent behavioral alterations.

39. CUMS increases DNA methylation at the GDNF promoter in both strains.
Goal
Investigate whether CUMS or IMI-induced alterations in the level of H3K27me3 and H3K4me3 at the GDNF promoter (Figures 2C and 2D) correlate with an increase in DNA methylation

40. CUMS increases DNA methylation at the GDNF promoter in both strains.
Findings
BALB mice
Increase in methylation at sites 2, 8-12 in HP compared to vSTR.
Significantly lower percentage methylated clones in vSTR in comparison to HP?
vSTR GDNF mRNA levels 13 fold higher then in HP
CUMS increases methylation at CpG sites 2 and 3. Reversed with IMI
B6 mice
CUMS increases methylation at CpG site 2 but not site 3 in the vSTR. Assumedly reversed with IMI.

41. CUMS increases DNA methylation at the GDNF promoter in both strains.

42. CUMS Increases the Binding of MeCP2 at the GDNF Promoter in Both Strains
Goal
Determine whether there is a difference in binding of MeCP2 to this promoter in the HP and vSTR of naïve adult BALB mice.
Findings
GDNF promoter containing DNA fragments were significantly less common in MeCP2 immunoprecipitates prepared from the vSTR compared with those from the HP

43. CUMS Increases the Binding of MeCP2 at the GDNF Promoter in Both Strains
Goal
See the effect of 6 weeks of CUMS and continuous IMI treatment on the binding of MeCP2 to the GDNF promoter
Findings
analysis revealed that CUMS significantly increased MeCP2 binding to the Gdnf promoter in both BALB and B6 mice, and continuous IMI treatment reversed this effect in stressed BALB mice

44. CUMS Increases the Binding of MeCP2 at the GDNF Promoter in Both Strains
Findings
The methylation of CpG site 2 is important for the genetic repression of GDNF expression

45. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice
Goal
Determine if the binding of MeCP2-HDAC2 complexes to the methylated CpG site of the GDNF promoter lead to decreased expression of GDNF after CUMS.
Determine the effect of CUMS on the binding of MeCP2-HDAC2 complexs at the GDNF promoter.

46. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice
Findings
CUMS increases the formation of MeCP2-HDAC2 complexes in stressed BALB mice. Reversed with IMI.
CUMS increases methylation at GDNF promoter-containing DNA fragments in BALB mice but not B6 Mice, when compared to non-stressed mice. Reversed with IMI

47. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice cont.
Goal
Investigate the role of DNA methylation in the CUMS-induced suppression of GDNF expression and on depression-like behaviors through application of zebularin (ZEB – DNA methyltransferase inhibitor)
See if RG-108 can reverse depressive behaviors in BALB mice

48. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice
ZEB Findings
Mice displayed increased social interaction time and increased sucrose preference
Decrease in latency to feeding in NSFT in stressed mice vs. non-stressed mice
In FST immobility time was significantly shorter in stressed and non-stressed mice
GDNF mRNA levels were greater then in control mice

49. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice
RG-108 findings
Increased social interaction time and sucrose preference in stressed mice

50. CUMS Increases the Binding of MeCP2-HDAC2 to the GDNF Promoter in BALB Mice
Also noted
CUMS led to an increase in mRNA expression for DNA methyltransferase 1 (DNMT1) and DNMT3a, but not DNMT3b, in the vSTR of stressed mice
This strongly suggests that that DNA methylation is critical for the CUMS induced GDNF repression and subsequent depression-like behaviors in BALB mice.

51. CUMS Increases Binding of MeCP2-CREB to the Gdnf Promoter in B6 Mice
Goals
Investigate whether the binding of the MeCP2-CREB complex to the GDNF promoter may be a causal mechanism of the increased GDNF expression in stressed B6 mice.
Investigate the binding of MeCP2-CREB complexes at the GDNF promoter

52. CUMS Increases Binding of MeCP2-CREB to the Gdnf Promoter in B6 Mice
Findings
No difference in the formation of MeCP2-CREB complexes between stressed and non stressed mice in both strains
GDNF promoter-containing DNA fragments of stressed B6 mice were significantly enriched in the reimmunoprecipitates of samples treated with CREB antibodies compared with those of nonstressed mice. This was not seen in stressed BALB mice
This suggest that CUMS induced binding of MeCP2-CREB complexes to the GDNF promoter leads to the activation of its transcription.

53. Discussion
The Data provide evidence that differential epigenetic marks in the NAc, along with environmental and genetic factors, may influence either the susceptibility or adaptation responses of an organism to chronic daily stressful events.

54. Role of GDNF in stress responses
Important points
Data as a whole supports the hypothesis that the mesolimbic dopamine system is involved in the formation of susceptibility and resistance responses to chronic stress
While IMI rescued GDNF expression in stressed mice, it also upregulated other neurotrophic factors in multiple brain regions. Cant say that GDNF alone was responsible for what we saw.

55. CUMS and Antidepressants Affect Histone Modifications in the GDNF Promoter
Important notes
Data suggests that hyperactive HDACs are involved in the reduction of GDNF expression and subsequent depression-like behaviors induced by CUMS
Reduced H3K4me3 level at the Gdnf promoter in the NAc may be a common mechanism for responses to CUMS, and the reduced H3K27me3 level may be one of the important mechanisms modulating the chromatin microenvironment that primes adaptation responses to CUMS.

56. DNA Methylation at the GDNF Gene Promoter Is Required for Both Susceptible and Adaptive Responses to CUMS
Important notes
Data indicates that CUMS enhances DNA methylation at particular CpG sites on the GDNF promoter in BALB mice.
Data suggests that the binding of different MeCP2 complexes (i.e., MeCP2-CREB and MeCP2-HDAC2) to the methylated CpG site on the GDNF promoter may be a causal mechanism for the induction and repression of GDNF expression in the NAc of B6 and BALB mice.

57. Conclusion
Dynamic epigenetic regulations of the GDNF promoter in the NAc play important roles in determining both the susceptibility and the adaptation responses to chronic stressful events.