FP04-1 Understanding Histone Deacetylase 9 (HDAC9) Function in GnRH Neuron Biology

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP04-GnRH & Gonadotroph Biology & Signaling
Saturday, June 15, 2013: 11:00 AM-11:30 AM
Presentation Start Time: 11:00 AM
Room 135 (Moscone Center)

Poster Board SAT-136
Smita Salian-Mehta*1, Mei Xu1, Connor Nash2, Mathew Scott Stratton3, Timothy A McKinsey3, Donald R Menick4, Stuart Allen Tobet5 and Margaret E Wierman6
1University of Colorado Denver, Aurora, CO, 2Colorado state university, Fort Collins, CO, 3University of Colorado Denver, 4Medical University of South Carolina, 5CO State Univ, Fort Collins, CO, 6University of Colorado Denver and Research Service VAMC, Aurora, CO
Abnormal sexual maturation and infertility in mice and humans result from disruption of Gonadotropin releasing hormone (GnRH) neuron development or function. Microarray analysis of GT1-7 (differentiated) as compared to NLT (undifferentiated) mouse GnRH neuronal cell lines revealed an upregulation of most HDACs and particularly class-IIa HDAC9. This class shuttles from cytoplasm to nucleus to classically act as transcriptional repressors although recent cytoplasmic action has been suggested during differentiation and development. Increases in HDAC9 by microarray (6-fold), RT-PCR (2.9-fold), Immunoblot (10-fold) and specific deacetylase activity in GT1-7 cells suggested that HDAC9 might play a role in GnRH neuronal development. Over-expression of hHDAC9 in NLT GnRH neuronal cells (low endogenous HDAC9) protected cells from serum withdrawal induced apoptosis as assessed by cleaved caspase 3 (0.6 vs.1.2 fold, compared to controls) and by Hoechst staining of condensed nuclei (0.5 fold), confirming a pro-survival role for HDAC9. Silencing of HDAC9 (by 80%) in GT1-7 cells, however, did not alter   caspase-3 cleavage, suggesting there may be redundancy with other Class II HDACs (4,5 and/or 7).The N-terminus of class-IIa HDACs is critical for protein-protein interactions (binding to transcription factors like MEF2) and C-terminus encodes the HDAC domain. To ask the role of the domains and nuclear versus cytoplasmic location to mediate HDAC9’s pro-survival effects, WT,  N- and C- terminal HDAC9 mutants were tested. Immunocytochemistry demonstrated WT hHDAC9 protein was expressed preferentially in the nucleus (N>C), whereas HDAC-N was only nuclear and HDAC9-C (deacetylase domain) was exclusively cytoplasmic. In response to growth factor withdrawal induced apoptosis,  both WT and C-terminal, but not N-terminal HDAC9 mutants showed decreased caspase 3/7 luminescence as compared to vector control (0.8-fold, p=0.05), suggesting that nuclear localization may not be critical for this cell specific effect. To ask if loss of HDAC9 had adverse effects on GnRH neuron development, GnRH neuron numbers were counted in brains of WT and HDAC9 null mice (E11-E16 based on crown-rump length). A 37% decrease in GnRH neurons was observed in HDAC9 null (210±82, n=5) compared to WT (335±87, n=5) embryonic brains. Thus our data supports the role of HDAC9 to promote neuron survival across GnRH neuronal development via a unique cytoplasmic rather than nuclear site of action.

Nothing to Disclose: SS, MX, CN, MSS, TAM, DRM, SAT, MEW

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: HD32119 to MEW
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