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

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

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

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.

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

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.

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

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.

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

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

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

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

Neurotrophic Factors cont.

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

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

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

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.

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.

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

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

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

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

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

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.

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

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.

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.

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

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.

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

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”

Role of HDAC2 in Behavioral Responses

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.

Role of HDAC2 in Behavioral Responses

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

Role of HDAC2 in Behavioral Responses

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

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.

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

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.

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

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

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

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

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.

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

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

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

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

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.

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

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.

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.

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.

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.

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.

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.