In vitro model of perimenopausal depression implicates steroid metabolic and proinflammatory genes

Sarah Rudzinskas, Jessica F Hoffman, Pedro Martinez, David R Rubinow, Peter J Schmidt, David Goldman, Sarah Rudzinskas, Jessica F Hoffman, Pedro Martinez, David R Rubinow, Peter J Schmidt, David Goldman

Abstract

The estimated 20-30% of women who develop perimenopausal depression (PMD) are at an increased risk of cardiovascular and all-cause mortality. The therapeutic benefits of estradiol (E2) and symptom-provoking effects of E2-withdrawal (E2-WD) suggest that a greater sensitivity to changes in E2 at the cellular level contribute to PMD. We developed an in vitro model of PMD with lymphoblastoid cell lines (LCLs) derived from participants of a prior E2-WD clinical study. LCLs from women with past PMD (n = 8) or control women (n = 9) were cultured in three experimental conditions: at vehicle baseline, during E2 treatment, and following E2-WD. Transcriptome analysis revealed significant differences in transcript expression in PMD in all experimental conditions, and significant overlap in genes that were changed in PMD regardless of experimental condition. Of these, chemokine CXCL10, previously linked to cardiovascular disease, was upregulated in women with PMD, but most so after E2-WD (p < 1.55 × 10-5). CYP7B1, an enzyme intrinsic to DHEA metabolism, was upregulated in PMD across experimental conditions (F(1,45) = 19.93, p < 0.0001). These transcripts were further validated via qRT-PCR. Gene networks dysregulated in PMD included inflammatory response, early/late E2-response, and cholesterol homeostasis. Our results provide evidence that differential behavioral responsivity to E2-WD in PMD reflects intrinsic differences in cellular gene expression. Genes such as CXCL10, CYP7B1, and corresponding proinflammatory and steroid biosynthetic gene networks, may represent biomarkers and molecular targets for intervention in PMD. Finally, this in vitro model allows for future investigations into the mechanisms of genes and gene networks involved in the vulnerability to, and consequences of, PMD.

Trial registration: ClinicalTrials.gov NCT00060736 NCT00001231.

Conflict of interest statement

The authors have no potential conflicts of interest or financial support regarding this manuscript.

© 2020. The Author(s), under exclusive licence to Springer Nature Limited.

Figures

Figure 1 –. PMD vs. Control LCLs:…
Figure 1 –. PMD vs. Control LCLs: in vitro estradiol (E2) exposures
LCLs were subjected to the following experimental treatment time course, creating three treatment groups: baseline, E2, and E2-withdrawal, per diagnostic category (control or PMD). 100nM E2 or vehicle (DMSO) was spiked into the cultures every 24 hours for a total of 72 hours of exposure, with an additional 24 hours of vehicle for LCLs in the E2-withdrawal treatment group. Two LCL cohorts were used: Cohort 1 was a smaller, subject-matched discovery cohort in which RNA-sequencing was performed with resulting differential gene expression (DEG) and gene network analysis, E2 quantification, and ELISAs on LCL culture media. Cohort 2 was an independent replication cohort that was used for qRT-PCR. For Cohort 1, all 8 PMD LCLs were derived from women with past PMD that was demonstrated to be E2-sensitive. For Cohort 2, all 18 PMD LCLs were derived from women with past PMD that underwent a similar psychiatric screening to the Cohort 1 PMD women but were not tested for E2-sensitivity. Thus, Cohort 2 represents an independent and broader PMD phenotype.
Figure 2 –. Significant Overlap between DEGs…
Figure 2 –. Significant Overlap between DEGs in all experimental treatment groups
Venn Diagrams indicating the overlap between genes that are differentially expressed (either up- or down- regulated) (p−41, Jaccard Exact Test). B) Overlap between transcripts that are upregulated (DEGs p−40, Jaccard Exact Test). C) Overlap between transcripts that are downregulated (DEGs p−31, Jaccard Exact Test).
Figure 3 –. CXCL10, a proinflammatory chemokine,…
Figure 3 –. CXCL10, a proinflammatory chemokine, is significantly increased in LCLs from women with PMD, particularly after E2-Withdrawal
A) Two-way ANOVA of LCL gene expression (RNA-seq); diagnosis x experimental treatment. CXCL10 expression significantly increased in PMD LCLs compared to controls (F1,45=15.82, p=0.0003), and in response to E2 treatment (F2,45=6.041, p=0.0047). Sidak posthoc t-tests show a significant increase specifically after E2-withdrawal (t45=4.06, p<0.0006, ***). B, C, D) qRT-PCR validation of RNAseq CXCL10 expression confirmed significant increases in transcript expression in PMD LCLs at baseline (t(37)=2.155, p=0.038, *), E2 (t(36)=2.046, p=0.048, *) and E2-withdrawal (t(37)=2.757, p<0.009, **). E, F) ELISA on the cell culture media on the day of LCL collection from a subset (n=6 controls, n=5 PMD) of the RNA-seq samples. Levels of the chemokine CXCL10 were significantly upregulated in the cell culture supernatant of LCLs from women with PMD compared to controls at baseline (t(9)=3.438, p=0.007, **) and after E2-withdrawal (t(9)=2.984, p=0.015, *).
Figure 4 –. CYP7B1 , the gene…
Figure 4 –. CYP7B1, the gene encoding the enzyme involved in DHEA metabolism, is significantly increased in LCLs from women with PMD, and significantly correlates to decreased plasma DHEA levels
A) A two-way ANOVA of gene expression (RNA-seq) demonstrated a significant main effect of diagnosis in PMD vs controls (F1,45=19.93, p<0.0001, ***), but showed no significant main effect specifically in response to E2-treatment (F2,45=0.358, p=0.701, ns), nor a significant interaction (F2,45=0.437, p=0.648, ns). B, C, D) qRT-PCR validation of the expression levels revealed that CYP7B1 was significantly increased at baseline (t(37)=2.345, p=0.025, *), as well as after E2-withdrawal (t(35)=2.378, p=0.023, *), and trended toward significance with E2 (t(35)=2.02, p=0.051, ns). E) Available plasma DHEA levels on a subset (n=7 control, n=7 PMD) of women whose LCLs were in the RNA-sequencing cohort revealed significantly decreased DHEA in PMD women (t(13)=3.322, p=0.006, **) F) CYP7B1 E2-withdrawal RPKMs (Reads Per Kilobase per Million mapped reads from RNA-seq) demonstrated a significant negative correlation to plasma DHEA levels (r2=0.46, p<0.008). Small black dots represent the 95% confidence intervals of the linear regression line.

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