37Glucocorticoids Reduce Astrocyte Numbers

cells reported that GR knockdown promoted cell prolif-eration, which is in contrast to our results (21). This discrepancy suggests that the relationship between GR

expression and cell proliferation may depend on cell type. The mechanisms by which GR regulates cell prolif-eration should be determined in future research.

PP

P

Fig. 7. Repeated adrenocorticotropic hormone (ACTH) administration decreased glucocorticoid receptor (GR) expression and the number of astrocytes in vivo. A, B: GR expression in the frontal cortex (A) and hippocampus (B) after 14 days of saline or ACTH administration. The bottom graphs show the quantified data of the GR/GAPDH ratio. n = 3. C: GFAP and GAPDH were detected by western blotting in the frontal cortex after 14 days of repeated ACTH administration. D: Neuronal nuclei (NeuN)- or GFAP-positive cells were stained by immunohistochemistry and their numbers calculated from 30 slices. E: Representative pic-tures show GFAP-positive cells in the prefrontal cortex of rats administered with saline or ACTH for 14 days. Scale bar = 50 μm.

Glial  Number  in  PFC  for  Mood  Disorders  15  

10  

5  

x106  

Glial  N

umbe

r  

37Glucocorticoids Reduce Astrocyte Numbers

cells reported that GR knockdown promoted cell prolif-eration, which is in contrast to our results (21). This discrepancy suggests that the relationship between GR

expression and cell proliferation may depend on cell type. The mechanisms by which GR regulates cell prolif-eration should be determined in future research.

PP

P

Fig. 7. Repeated adrenocorticotropic hormone (ACTH) administration decreased glucocorticoid receptor (GR) expression and the number of astrocytes in vivo. A, B: GR expression in the frontal cortex (A) and hippocampus (B) after 14 days of saline or ACTH administration. The bottom graphs show the quantified data of the GR/GAPDH ratio. n = 3. C: GFAP and GAPDH were detected by western blotting in the frontal cortex after 14 days of repeated ACTH administration. D: Neuronal nuclei (NeuN)- or GFAP-positive cells were stained by immunohistochemistry and their numbers calculated from 30 slices. E: Representative pic-tures show GFAP-positive cells in the prefrontal cortex of rats administered with saline or ACTH for 14 days. Scale bar = 50 μm.

Chronic Stress as a Potential Non-Genetic Cause for Bipolar Disorder Anant Naik1, Bhavani Murakonda1, Atsushi Asakura2

1Department of Biomedical Engineering, University of Minnesota Twin Cities, 2Department of Neurology, University of Minnesota Twin Cities

A.

Figure 3: Patients with bipolar disorder have a significant decline in glia in the dlPFC. A) Using obtained brain slices, Ongur et al. looked at Agranular area 24 in the dlPFC to ascertain glial density. This region has been shaded. B) A comparison by Ongur et al. of patients with various mood disorders, and the glial number found. Patients with BD, indicated by fBD (familial) and oBD (other) have a decreased number of glial cells (p<0.05). The decrease in fBD is substantially lower than other mood disorders. (Ongur et al., 1998, PNAS)

References 1.  Aas, M., et al. Additive effects of childhood abuse and cannabis abuse on clinical

expressions of bipolar disorders. Psychological medicine 44.08 (2014): 1653-1662. 2.  Aas, Monica, et al. The role of childhood trauma in bipolar disorders. International

journal of bipolar disorders 4.1 (2016): 1-10. 3.  Bipolar Disorder Among Adults. NIMH. n.d. 4.  Brooks, John O., et al. Preliminary evidence of differential relations between

prefrontal cortex metabolism and sustained attention in depressed adults with bipolar disorder and healthy controls. Bipolar disorders 8.3 (2006): 248-254.

5.  Cerqueira, Joao J., et al. Morphological correlates of corticosteroid-induced changes in prefrontal cortex-dependent behaviors. The Journal of Neuroscience 25.34 (2005): 7792-7800.

6.  Cerqueira, João J., et al. The prefrontal cortex as a key target of the maladaptive response to stress. The Journal of Neuroscience 27.11 (2007): 2781-2787.

7.  Craddock, Nick, and Pamela Sklar. Genetics of bipolar disorder. The Lancet 381.9878 (2013): 1654-1662.

8.  Grande, Iria, et al. Bipolar disorder. The Lancet (2015). 9.  Öngür, Dost, Wayne C. Drevets, and Joseph L. Price. Glial reduction in the

subgenual prefrontal cortex in mood disorders. Proceedings of the National Academy of Sciences 95.22 (1998): 13290-13295.

10. Otto MW, Perlman CA, Wernicke R, Reese HE, Bauer MS, Pollack MH. Posttraumatic stress disorder in patients with bipolar disorder: a review of prevalence, correlates, and treatment strategies. Bipolar Disord 2004: 6: 470–479.

11. Pompili, Maurizio, et al. Epidemiology of suicide in bipolar disorders: a systematic review of the literature. Bipolar disorders 15.5 (2013): 457-490.

12. Rajkowska, Grazyna, Angelos Halaris, and Lynn D. Selemon. Reductions in neuronal and glial density characterize the dorsolateral prefrontal cortex in bipolar disorder. Biological psychiatry 49.9 (2001): 741-752.

13. Salzbrenner, Stephen, and Eileen Conaway. Misdiagnosed Bipolar Disorder Reveals Itself to be Posttraumatic Stress Disorder with Comorbid Pseudotumor Cerebri: A Case Report. Psychiatry (Edgmont) 6.8 (2009): 29.

14. Unemura, Kazuhiro, et al. Glucocorticoids decrease astrocyte numbers by reducing glucocorticoid receptor expression in vitro and in vivo. Journal of pharmacological sciences 119.1 (2012): 30-39.

15. Vermeer, Harry, et al. An in vitro bioassay to determine individual sensitivity to glucocorticoids: induction of FKBP51 mRNA in peripheral blood mononuclear cells. Molecular and cellular endocrinology 218.1 (2004): 49-55.

Hypothesis Abstract

Background

Rationale

Stress Disorders are Associated with BD

Astrocyte Densities Decline with GC in vitro

Adverse Impact of Stress on Neurons in vivo

Significance & Innovation

Sample  Characteris7cs Sample  Size Rate  of  PTSD% Bipolar patients admitted for mania or mixed 71 17

National general population survey: respondents with bipolar I characterized by euphoria, grandiosity, and excessive energy

29 39

Inpatient and outpatient bipolar patients 50 40

Bipolar patients, manic or mixed: first admission for psychosis 77 21

Bipolar I and II outpatients recruited from community 288 7

Bipolar patients: first admission for psychosis 102 11

Bipolar I and II, treatment-seeking outpatients in the Systematic Treatment Enhancement Program for bipolar disorder

475 17

Bipolar I and II, treatment-seeking outpatients 122 19

Figure 5: A representation from Cerquiera et. al. demonstrating the decline in neuronal number in the medial PFC in mice exposed to chronic stress. A) Volumetric decline in neurons shown in Layer I, II, III-VI, and molecular (Mol) and pyramidal (Pyr) layers of the subiculum of the cortex. Dramatic and statistically significant decline is evident in Layer I & II (*P < 0.05, **P < 0.01) B) Unemura et al. shows a decline in average glial densities in both Frontal Cortex and Hippocampus after administration of Adrenocorticotropic Hormone (ACTH) in vivo, immunostaining using GFAP. Scale bar = 50 microns. (Cerqueira et al., 2007, J Neurosci) C) A pilot study done by Unemura et al presented showing the decline of aggregate GFAP+ astrocytes in the frontal cortex and hippocampus post-ACTH treatment (P < 0.05) (Unemura et al., 2012, J Phar Sci.)

Chronic stress-induced GC elevation in the dlPFC via the HPA axis results in a decline in GCRs and astrocyte death in the dlPFC. BD patients experienced a decline in neuronal metabolic activity in the PFC, which is physiologically associated with the decline in astrocyte activity. Because of this, we hypothesize the effects of chronic stress on the dlPFC is a potential non-genetic cause for BD.

Bipolar disorder (BD) is a neuroprogressive disorder which is characterized by alternating episodes of depression and mania (12). It can last the lifetime of the patient, leading to a decreased quality of life and high burden of disease. Current estimates suggest that it affects around 2.6% of the national population, however diagnosis is complicated and likely under diagnosed. Currently the diagnostic criteria includes at least one manic episode for Bipolar I disorder, while it is at least one hypomanic episode and one major depressive episode for Bipolar II disorder (8). The average delay between the onset of BD in a person and the diagnosis is approximately 5-10 years (8). This delay can lead to severe consequences because people with BD are at a risk of committing suicide 20-30 times greater than that for the general population (11). While lithium and valproate are commonly used in order to stabilize the mood of the patients, they do not offer a permanent solution to the disease. This is largely because the underlying cause of the disorder is still unknown and therefore a curative treatment is yet to be discovered. Research has found that there is a major genetic aspect to the disorder (7), but non-genetic causes have not been ruled out. Having a better understanding in either aspect could lead to better management and a possible treatment of the disorder.

The proposed hypothesis provides a possible explanation for why patients who experience incredible stress develop BD compared to a baseline level. The verification of our hypothesis would tremendously expand treatment options for many patients, particularly veterans and abuse victims, because more thorough psychiatric evaluations would be emphasized over genetic pedigree analysis. Our hypothesis also begs further questions regarding BD’s relationship with PTSD, along with other psychiatric disorders. This hypothesis would show that there is a direct link between chronic stress and BD. This would allow for possible treatments or ways to prevent it before the onset. Additionally, the treatment would focus on easing stress rather than the stabilization of the mood. This hypothesis can also open the doors to a more efficient way to diagnose BD in its early stages. Using the principles of regenerative medicine, including genetic transfection and stem cell therapy, researchers can effectively test our hypothesis that BD is induced by chronic stress. Given our hypothesis, the implantation of glial stem cells in vivo into the dlPFC would provide a novel treatment to BD. These experiments would prevent misdiagnoses of patients of sexual abuse, trauma, and PTSD with BD without any discernable family history, which directly impacts patient care, and the quality of life for people with disease.

A.

B.

34 K Unemura et al

results suggest that glucocorticoids inhibited astrocyte proliferation and reduced GR expression mediated via GR.

GR knockdown led to a reduction in astrocyte numbersTo investigate the relationship between reduction in

GR expression and inhibition of astrocyte proliferation by glucocorticoids, siRNA targeting GR was transfected

Fig. 2. Corticosterone (CRT) and dexamethasone (DEX) reduced the number of astrocytes via a glucocorticoid receptor but did not induce astrocytic damage. A, B: The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was per-formed 72 h after treatment with CRT (0.01 – 1 μM) and DEX (0.01 – 1 μM). C: The lactate dehydrogenase (LDH) assay was performed 72 h after treatment with CRT (1 μM) and DEX (1 μM) or 24 h after treatment with H2O2 (1 mM). D, E, F: The MTT assay was performed 72 h after treatment with CRT (1 μM); DEX (1 μM); RU486 (0.3 – 3 μM), a GR antagonist; and eplerenone (10 μM), a mineralcorticoid receptor antagonist. n = 4. ***P < 0.001 vs. control, ###P < 0.001 vs. CRT or DEX alone. n.s., not significant.

Fig. 3. Reduction in astrocyte prolif-eration by corticosterone (CRT) and dexamethasone (DEX) via a glucocor-ticoid receptor. Bromodeoxyuridine (BrdU)- and GFAP-positive cells were detected 48 h after treatment with CRT (1 μM), DEX (1 μM), and RU486 (3 μM) plus BrdU (1 μM). A: CRT, B: DEX. n = 8. **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. CRT or DEX alone.

34 K Unemura et al

results suggest that glucocorticoids inhibited astrocyte proliferation and reduced GR expression mediated via GR.

GR knockdown led to a reduction in astrocyte numbersTo investigate the relationship between reduction in

GR expression and inhibition of astrocyte proliferation by glucocorticoids, siRNA targeting GR was transfected

Fig. 2. Corticosterone (CRT) and dexamethasone (DEX) reduced the number of astrocytes via a glucocorticoid receptor but did not induce astrocytic damage. A, B: The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was per-formed 72 h after treatment with CRT (0.01 – 1 μM) and DEX (0.01 – 1 μM). C: The lactate dehydrogenase (LDH) assay was performed 72 h after treatment with CRT (1 μM) and DEX (1 μM) or 24 h after treatment with H2O2 (1 mM). D, E, F: The MTT assay was performed 72 h after treatment with CRT (1 μM); DEX (1 μM); RU486 (0.3 – 3 μM), a GR antagonist; and eplerenone (10 μM), a mineralcorticoid receptor antagonist. n = 4. ***P < 0.001 vs. control, ###P < 0.001 vs. CRT or DEX alone. n.s., not significant.

Fig. 3. Reduction in astrocyte prolif-eration by corticosterone (CRT) and dexamethasone (DEX) via a glucocor-ticoid receptor. Bromodeoxyuridine (BrdU)- and GFAP-positive cells were detected 48 h after treatment with CRT (1 μM), DEX (1 μM), and RU486 (3 μM) plus BrdU (1 μM). A: CRT, B: DEX. n = 8. **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. CRT or DEX alone.

36 K Unemura et al

These results suggest that secretion of high concentra-tions of glucocorticoids induced by repeated ACTH ad-ministration leads to a reduction in both GR expression and the number of astrocytes in vivo.

Discussion

This study investigated the mechanism by which glu-cocorticoids inhibit astrocyte proliferation. The results demonstrated a relationship between astrocyte prolifera-tion and GR expression following treatment with gluco-corticoids. This was demonstrated in an animal model (in vivo) as well as cultured astrocytes (in vitro). In sum-mary, our results suggest that glucocorticoids reduce astrocyte proliferation by inducing a reduction in GR expression.

In the first experiment, the subcellular localization of GR was observed in cultured astrocytes, with GR found to be primarily expressed in the nuclei (Fig. 1). Neuronal GR performs functions other than gene transcription, for example, GR interacts with receptor tyrosine kinase for BDNF (trkB) and promotes glutamate release (20). In contrast, functions of astrocytic GR other than the func-tion of binding to a glucocorticoid have not yet been re-ported. Based on our results, we assumed that astrocytic as well as neuronal GRs have some unknown functions in our culture conditions.

Corticosterone and dexamethasone reduced the num-ber of astrocytes via GR by inhibiting astrocyte prolifera-

tion (Figs. 2 and 3). These results are consistent with those of a previous report that suggested that the neural cell adhesion molecule (NCAM) inhibited astrocyte proliferation via GR (16). In neurons and astrocytes, GR expression has also been shown to change with glucocor-ticoid administration in vitro (19). The mechanisms by which GR expression affects astrocyte function are largely unknown. We showed that short-term (1 – 6 h) glucocorticoid treatment increased GR expression, whereas long-term (> 12 h) treatment decreased GR ex-pression. RU486 also increased GR expression in a short term; however, this expression did not decrease subse-quently (Fig. 4). We examined the effect of RU486 on the glucocorticoid-induced change in GR expression. GR expression increased by glucocorticoid treatment was enhanced by RU486, whereas GR expression reduced by glucocorticoid treatment was inhibited by RU486 (Fig. 5).

We hypothesized that reduced GR expression follow-ing long-term glucocorticoid treatment plays a crucial role in astrocyte proliferation. To validate this hypothesis, we used siRNA to silence GR. GR knockdown reduced the number of astrocytes by inhibiting their proliferation (Fig. 6). GR antagonism by RU486 did not induce pro-motion of astrocyte proliferation. We suppose that this is because RU486 does not decrease GR expression in as-trocyte. Thus, we propose that prolonged reduction in GR expression inhibits proliferation of cultured astro-cytes. However, another study using murine macrophage

Fig. 6. Glucocorticoid receptor (GR) knockdown reduced astrocyte prolifera-tion. A: Representative western blotting example of GR and GAPDH 96 h after siRNA transfection. B: Quantification of the GR/GAPDH ratio in panel A. n = 4. C: The MTT assay was performed 120 h after siRNA transfection. n = 4. D: BrdU-positive cells were detected 48 h after BrdU treatment (1 μM) 72 h after siRNA transfection. n = 8. *P < 0.05, ***P < 0.001 vs. control siRNA.

Figure 4: A summary of the results from Unemura et al. showing a decline in astrocyte density in vitro. A) A decline of functionality in astrocytes is evident with greater concentrations of cortisone compared to control (*** P < 0.05) B) Application of cortisone on astrocytes dramatically reduced cell number compared to control (*** P < 0.05). C) Cortisone application decreases prevalence of Glucocorticoid Receptor (GR) RNA, an indicator for the prevalence of GR in cells (*P < 0.05). (Unemura et al., 2012, J Phar Sci.)

A. B. C.

Figure 3: A table describing results from Otto et al. A high percentage of patients with BD were found to also exhibit PTSD. The National Population Survey with BD I shows 39% prevalence, in addition to Inpatient and outpatient BD patients. Other numbers also demonstrate the comorbidity. (Otto et al., 2004, Bipolar Disorder)

Bipolar disorder (BD) is a disorder characterized by alternating episodes of depression and mania. Present treatments are aimed at stabilizing the mood of the patients and do not offer a permanent solution to the disease. While research has found that there is a major genetic aspect to the disorder, non-genetic causes have not been thoroughly explored. Having a better understanding in either aspect could lead to better management and a possible treatment of the disorder. We hypothesize that chronic stress-induced glucocorticoid (GC) elevation in the dorsolateral prefrontal cortex (dlPFC) via the Hippocampal-Pituitary-Adrenal (HPA) axis results in a decline in GC Receptors and astrocyte death in the dlPFC, causing BD. In vivo and in vitro studies have confirmed that an elevation in GC has decreased astrocyte densities and declined functionality, but have not compared them to levels exhibited by BD patients. This discovery would allow for better diagnosis of BD in patients that don’t exhibit a pedigree of BD history. Using the important tools of regenerative medicine, such as genetic transfection and stem cell therapy, researchers can effectively test this hypothesis, leading to important novel treatments for a disease that affects millions of people.

B.

NeuronAstrocyte

Blood vessel

GC

Neuron

Blood vessel

GCDegeneration of Astrocyte

In Prefrontal Cortex

Bipolar Disorder

Figure 2: A visual representation of the development of BD induced by stress. A) Chronic stress results in an upregulation of the HPA axis, resulting in the hyperactivity of the adrenal gland. This creates the adverse upregulation of glucocorticoids in the blood, which specifically targets the dlPFC, a phenomenon less understood. B) The upregulation of glucocorticoids in the blood stream causes astrocyte death in the dlPFC, which induces the effects of BD.

A. B. Cerebrum and Cerebellum Prefrontal Cortex

Endocrine GC Feedback to

stress

Endocrine response to

stress

Adrenal Gland

GC release due to stress

PFC

Stress Hypothalamus

Bipolar Disorder

Blood Vessel

Blood Vessel

Neuron

Neuron

Astrocyte

Degeneration of Astrocyte

Figure 1: The progression of BD in the life of a patient. Transient oscillations of hypomania/mania and depression are evident throughout a patient’s life. Age of onset is in early adolescence (Grande et al., 2015, The Lancet)

Life Progression of Bipolar Disorder Mania

Hypomania

Euthymia

Subthreshold Depression

Major Depression

Mixed State

Sev

erity

of

Man

ia

Sev

erity

of

Dep

ress

ion

C.

BD patients have Glial Deficits in the dlPFC

Cor7sone  Impact  on  Astrocyte  Func7onality  

Cor7sone  Impact  on  Astrocyte  Number  

Cor7sone  Impact  on  GR  RNA  Prevalence  

Acknowledgments Our work could not have been completed without the support of Dr. Asakura, and input from Dr. Russell Carter in the Department of Neuroscience, University of Minnesota.

Volume  of  Neurons  in  the  mPFC  Pre/Post-­‐Stress  

ACTH  impact  on  Astrocytes  in  vivo  

Saline                            ACTH