A Dominant Negative ERb Splice Variant Determines the Effectiveness of Early or Late Estrogen Therapy after Ovariectomy in Rats Jun Ming Wang1,2,3,4*, Xu Hou 3 , Samuel Adeosun 3 , Rosanne Hill1, Sherry Henry1, Ian Paul2, Ronald W. Irwin7, Xiao-Ming Ou2, Steven Bigler1, Craig Stockmeier2, Roberta Diaz Brinton7, Elise Gomez-Sanchez5,6 1 Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 2 Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 3 Program in Neuroscience, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 4 Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 5 GV (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 6 Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 7 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, United States of America Abstract The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) b variant, ERb2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERb2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERb2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERb2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERb2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERb2 in circulating white blood cells and brain cells suggests that ERb2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain. Citation: Wang JM, Hou X, Adeosun S, Hill R, Henry S, et al. (2012) A Dominant Negative ERb Splice Variant Determines the Effectiveness of Early or Late Estrogen Therapy after Ovariectomy in Rats. PLoS ONE 7(3): e33493. doi:10.1371/journal.pone.0033493 Editor: Susan Kovats, Oklahoma Medical Research Foundation, United States of America Received September 6, 2011; Accepted February 14, 2012; Published March 13, 2012 Copyright: 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by an Alzheimer’s Association Investigator Initiated Research Grant (133086), a training grant from a center grant from the National Center for Research Resources (NCRR, RR17701), a component of NIH, and an Intramural Research Support Program grant from University Mississippi Medical Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jwang@umc.edu Introduction Most women in developed countries now live 25 to 30 years, one third of their lives, after menopause, during which time they are at increased risk for cardiovascular disease, osteoporosis and dementia. Research into the etiology of menopausal dementia leading to the development of effective prevention and therapeutic strategies, will thereby improve women’s overall health and quality of life, and significantly reduce the public healthcare burden. Basic science studies and observational trials [1–6] suggested that estrogen therapy (ET) would prevent cognitive decline associated with menopause, however, this was challenged by the results from the Women’s Health Initiative and the ancillary Memory Study (WHIMS) in 2002 [7,8]. Among reasons cited for the discrepancy were differences in the type of estrogenic compounds used, routes of administration, cyclic versus continu- ous regimens, concomitant use of progestins, and the time after menopause that the ET was initiated. The latter led to the hypothesis of a critical period, or therapeutic window, during which ET must be initiated to obtain maximum benefit [1,5,9,10]. To date, the molecular basis of this hypothesis and the boundaries of the clinical therapeutic window are unknown. Many of the effects of estrogens are mediated by estrogen receptors (ER), primarily ERa and ERb, which are encoded by different genes. Both ERa and ERb are expressed in the brain. ERb is thought to be more important in sleep disturbance, irritability, depression and anxiety, panic disorders, and cognitive dysfunction [11–16]. The ERb transcripts are spliced into different mRNAs and translated into ERb variants with different biological functions depending physiological or pathological conditions. One of these, hERb2/hERbcx found in humans and non-human primates, contains unique 26 amino acid (aa) residues which replace the aa residues encoded from exon 8 in the C-terminal part of the ligand binding domain. hERb2 forms dimers with other ER, but is unable to bind ligands or coactivators and has no transcriptional activity in reporter assays[17,18]. Ectopic expres- PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33493