. We propose that there is a biological switch in estrogen receptor action that occurs coincident with age and length of time after ovarian hormone depletion (i.e. menopause). Accumulating evidence suggests that ERb shifts from a predominantly ligand-activated transcription factor (major role during reproductive years) to a ligand-independent transcription factor post-menopause. Elucidating the molecular basis for this shift in signaling paradigms is critical for understanding clinical observations, which demonstrate a defined narrow window of time for therapeutic efficacy of hormone therapy in postmenopausal women. This application is a competitive renewal of R01 AG033605. Our previous aims for this project centered on factors that we predicted would differentially dictate a dominant ligand-dependent signaling pattern for ERb, as opposed to a ligand-independent signaling pattern. Through that work, we identified 3 key molecular factors that facilitated ligand-independent function of ERb in the aged brain: receptor phosphorylation, alternative RNA splicing, and coregulatory protein interactions. These mechanisms represented both direct changes to the receptor itself and indirect changes in protein:protein interactions. The experiments proposed in this renewal will be focused on the direct changes to the ERb protein itself, namely phosphorylation and alternative splicing.
Aim 1 will focus on posttranslational phosphorylation of the receptor. Phosphorylation of ERb is a strong facilitator of ligand-independent activity, yet the extent of phosphorylated ERb present in the aged female brain, and the in vivo functional consequences of such a modification, represents a major gap in our current knowledge. We propose a comprehensive proteomics approach to a) quantify phosphor-ERb in the aged female brain, b) determine how site-specific phosphorylation alters ERb protein:protein interactions, and c) identify specific phospho-ERb target genes.
Aim 2 will focus on alternative RNA splicing of the nascent ERb transcript. Our data and others have demonstrated the functional significance of ERb splice variants and we have shown that these alternative variants increase during aging. However, we lack a fundamental understanding of the molecular mechanisms regulating ERb splicing in the brain, or in any tissue type. We have now identified several putative splicing factors that regulate alternative splicing of ERb in the aged brain.
This aim will test the direct effects of these splicing factors on ERb alternative splicing and also determine if E2 regulates global splicing events by modulating the expression of these splicing factors. Our preliminary data also showed that transcriptional kinetics play an important role in ERb splicing, with slower transcription favoring exon inclusion resulting in increased ERb2.
This aim will further explore this relationship to test how transcriptional kinetics in the brain change in our aging female rat menopause model. Impact: Understanding the basic molecular signaling pathways of E2 in the aging brain will help drive therapeutic advances and inform treatment strategies for postmenopausal women.

Public Health Relevance

The positive health benefits of estrogen replacement therapy in the aged brain have gained considerable interest in recent years, however our understanding of the molecular mechanisms regulating estrogen signaling in the aged brain is limited. This proposal will investigate the mechanisms regulating direct molecular modifications to the estrogen receptor, which ultimately alter the efficacy of the receptor to mediate estrogen signaling pathways and gene targets in the brain.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG033605-07
Application #
9547751
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mackiewicz, Miroslaw
Project Start
2009-08-01
Project End
2022-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Loyola University Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
Shults, Cody L; Dingwall, Caitlin B; Kim, Chun K et al. (2018) 17?-estradiol regulates the RNA-binding protein Nova1, which then regulates the alternative splicing of estrogen receptor ? in the aging female rat brain. Neurobiol Aging 61:13-22
Asimes, AnnaDorothea; Kim, Chun K; Cuarenta, Amelia et al. (2018) Binge Drinking and Intergenerational Implications: Parental Preconception Alcohol Impacts Offspring Development in Rats. J Endocr Soc 2:672-686
Kim, C K; Torcaso, A; Asimes, A et al. (2018) Structural and functional characteristics of oestrogen receptor ? splice variants: Implications for the ageing brain. J Neuroendocrinol 30:
Pinceti, Elena; Shults, Cody L; Rao, Yathindar S et al. (2016) Differential Effects of E2 on MAPK Activity in the Brain and Heart of Aged Female Rats. PLoS One 11:e0160276
Rao, Yathindar S; Pak, Toni R (2016) microRNAs and the adolescent brain: Filling the knowledge gap. Neurosci Biobehav Rev 70:313-322
Rao, Yathindar S; Shults, Cody L; Pinceti, Elena et al. (2015) Prolonged ovarian hormone deprivation alters the effects of 17?-estradiol on microRNA expression in the aged female rat hypothalamus. Oncotarget 6:36965-83
Shults, Cody L; Pinceti, Elena; Rao, Yathindar S et al. (2015) Aging and Loss of Circulating 17?-Estradiol Alters the Alternative Splicing of ER? in the Female Rat Brain. Endocrinology 156:4187-99
Pinceti, E; Shults, C L; Rao, Y S et al. (2015) Phosphorylation Alters Oestrogen Receptor ?-Mediated Transcription in Neurones. J Neuroendocrinol 27:861-71
Mott, Natasha N; Pinceti, Elena; Rao, Yathindar S et al. (2014) Age-dependent Effects of 17?-estradiol on the dynamics of estrogen receptor ? (ER?) protein-protein interactions in the ventral hippocampus. Mol Cell Proteomics 13:760-79
Tajuddin, Nuzhath F; Przybycien-Szymanska, Magdalena M; Pak, Toni R et al. (2013) Effect of repetitive daily ethanol intoxication on adult rat brain: significant changes in phospholipase A2 enzyme levels in association with increased PARP-1 indicate neuroinflammatory pathway activation. Alcohol 47:39-45

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