Chronic stress alters local estradiol expression across brain regions in a sex-dependent way

Elliot Smith*Lizzy Hanson, Jessica Judd, Dylan Peay, Amanda Acuna, Bryce Badaruddin, Megan Donnay, Jinah Kim, Cindy Reynolds, Gillian Thornton, Chris Willis, Hovhannes Saribekyan

Cheryl Conrad1, PhD; Clark Presson1, PhD; Bob Gibbs2, PhD1Arizona State University, College of Liberal Arts & Sciences, Department of Psychology, 2University of Pittsburgh, School of Pharmacy

Why studying chronic stress and estradiol in rodents can inform us about Depression

Mood disorder• Persistent sadness• Loss of interest (anhedonia)

Global Disease Burden and Impact [1]• 300 million people suffer globally• Significant risk of death by suicide• Most patients fail to improve from existing treatments

• Transitions involving estrogen fluctuations (puberty, menses, pregnancy, menopause) contribute to MDD risk in women• Estradiol (E2): specific type of estrogen

• Suggests that estrogen may be play a role in MDD risk

Approximately twice as many women as men suffer from MDD [2]

Chronic stress (STR) in rodents can model some aspects of MDD symptoms and pathophysiology MDD and STR change the brain

in region-specific and sex-specific ways

Synapse remodeling in the HIPP• Inhibiting ARO-L (via letrozole), thus blocking E2 production, decreases HIPP dendritic

complexity [5]• These changes can be rescued by administration of E2 [10]

Behavioral consequences of inhibiting local E2 production• Both male and female ArKO (Aromatase-deficient) mice show worse HIPP- and PFC-

dependent short-term spatial memory than wild-type mice [6]• Female ArKO mice show higher depressive-like behavior than wild-type [4]

• Aromatase activity in women affects memory and cognition• Hormone-based breast cancer chemotherapy that inhibits aromatase (such as

letrozole) can impair memory • Future directions: region-specific hormonal mechanisms underlying MDD,

especially local E2 production via ARO-L• Measuring brain and serum E2 and T

Why Major Depressive Disorder (MDD) is a significant problem

E2 & cholesterol protect against stress hormone-induced hippocampal neuronal dendritic retraction of castrated male and female rats [3,8]

These findings suggest that E2 may protect against chronic stress effects through an unknown mechanism• Are steroids locally produced in the brain, as gonads were

removed and could not produce E2 and other steroids?Lack of E2 protection may lead to heightened inflammatory response

The goal was to determine how chronic stress and sex affect local E2, ARO-L, and inflammatory expression across key brain regions

Method: Timeline and Processing

Stress Biomarkers: chronic stress reduced body weight gain, but did not alter adrenal size

Bodyweight Adrenals

Thymus

Chronic stress decreased thymus weights in males but not females.

** p < 0.01, # p < 0.05

Chronic stress reduced TNK-a (in the mPFC, cerebellum=CB) and IL-b (in CB),

** p < 0.01, # p < 0.05

Inflammatory Cytokines

Conclusions and Implications

References

HIP

P

Overview of Findings

MDD may be associated with altered local E2 synthesis across brain regions

Chronic stress may alter E2 production WITHIN the brain

~ not statistically significant

ARO-L mRNA expression

Goal 2: E2 Expressionn.c. no change

Goal 1: Stress and Inflammatory Biomarkers

Local E2 synthesis in the brain may be a critical mechanism for structural and functional changes in response to chronic stress

1) American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (DSM-5®). American Psychiatric Pub.Brown, S. M., Henning, S., & Wellman, C. L. (2005). Mild, short-term stress alters dendritic morphology in rat medial prefrontal cortex. Cerebral Cortex, 15(11), 1714-1722.

2) Albert, P. R. (2015). Why is depression more prevalent in women? Journal of psychiatry & neuroscience: JPN, 40(4), 219.3) Conrad, C. D., McLaughlin, K. J., Huynh, T. N., El-Ashmawy, M., & Sparks, M. (2012). Chronic stress and a cyclic regimen of estradiol administration separately facilitate spatial memory:

relationship with hippocampal CA1 spine density and dendritic complexity. Behavioral neuroscience, 126(1), 142.4) Dalla, C., Antoniou, K., Papadopoulou-Daifoti, Z., Balthazart, J., & Bakker, J. (2004). Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit ‘depressive-like’symptomatology.

European Journal of Neuroscience, 20(1), 217-228.5) Kretz, O., Fester, L., Wehrenberg, U., Zhou, L., Brauckmann, S., Zhao, S., ... & Rune, G. M. (2004). Hippocampal synapses depend on hippocampal estrogen synthesis. Journal of Neuroscience,

24(26), 5913-5921.6) Martin, S., Jones, M., Simpson, E., & van den Buuse, M. (2003). Impaired spatial reference memory in aromatase-deficient (ArKO) mice. Neuroreport, 14(15), 1979-1982.7) McLaughlin, K. J., Gomez, J. L., Baran, S. E., & Conrad, C. D. (2007). The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms.

Brain research, 1161, 56-64.8) Ortiz, J. B., McLaughlin, K. J., Hamilton, G. F., Baran, S. E., Campbell, A. N., & Conrad, C. D. (2013). Cholesterol and perhaps estradiol protect against corticosterone-induced hippocampal

CA3 dendritic retraction in gonadectomized female and male rats. Neuroscience, 246, 409-421.9) Prange-Kiel, J., Wehrenberg, U., Jarry, H., & Rune, G. M. (2003). Para/autocrine regulation of estrogen receptors in hippocampal neurons. Hippocampus, 13(2), 226-234.10)Zhou, Q. G., Hu, Y., Hua, Y., Hu, M., Luo, C. X., Han, X., ... & Zhu, D. Y. (2007). Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal

neurogenesis. Journal of neurochemistry, 103(5), 1843-1854.

Dendritic atrophy

Male rats likely showed greater bodyweight changes than females due to males being heavier. Stress did not alter adrenal size for either sex. ** p < 0.01, *** p < 0.001

Inflammatory Biomarkers: chronic stress reduced thymus weight in males and altered cytokines in both sexes

Brain Expression of E2: chronic stress may lead to region-specific sex differences in E2 expression

Chronic stress impacted males and females by reducing body weight gain and inflammatory cytokines, and also by reducing thymus weight.

Chronic stress tended to increase E2 expression in the male HIPP, female mPFC, and AMYG for males and females, and decrease E2 expression in the male mPFC.

• The brain contains enzymes to convert cholesterol into T and E2 [9]

• ARO-L may be a potential COMPENSATORY mechanism in the HIPPo Males showed higher hippocampal ARO-L expression than did females and a

pattern of chronic stress increasing ARO-L vs. control maleso Given that chronic stress leads to hippocampal dendritic retraction in males,

HIPP ARO-L increases in males are unlikely to be protective

ARO-L expression may be key in understanding sex-specific brain responses to chronic stress: ARO-L is necessary to convert T into E2

• ARO-L may be a potential PROTECTIVE mechanism in the mPFCo A pattern was observed with males showing decreased ARO-L mPFC

expression, while females showing increased ARO-L expression.o The mPFC is highly sensitive to chronic stress, undergoing dendritic changes

before the HIPP

Will need to test by blocking E2 synthesis to confirm this hypothesis

MDD symptoms

Brain regions of interest include the HIPP, mPFC, AMYG (where STR has been found to induce dendritic changes), and CB (where no STR-induced changes are expected)

Half of tissue lost due to shipping problems

Due to tissue loss, only patterns can be inferred. HIPP: STR tended to increase E2 in males, decrease E2 in females. AMYG: STR tended to increase E2 in both males and females.

mPFC: STR tended to decrease ARO-L in males, but not in females. AMYG: females tended to have higher ARO-L than did males. CB: expression pattern appeared similar regardless of stress or sex.

HIPP: ARO-L mRNA was lower in females than in males.* p < 0.05 Chronic stress tended to increase ARO-L of both sexes. # p < 0.05

E2 levels

Will need to castrate males and females to confirm this hypothesis

= ARO-L

= T

mP

FC