June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Overexpression of histone deacetylase 3 (HDAC3) induces apoptosis in differentiated retinal neurons
Author Affiliations & Notes
  • Heather M Schmitt
    Ophthalmology, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Cassandra Schlamp
    Ophthalmology, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Huan Yang
    Surgery, UW-Madison, Madison, Wisconsin, United States
  • Lian-Wang Guo
    Surgery, UW-Madison, Madison, Wisconsin, United States
  • Robert W Nickells
    Ophthalmology, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Heather Schmitt, None; Cassandra Schlamp, None; Huan Yang, None; Lian-Wang Guo, None; Robert Nickells, None
  • Footnotes
    Support  NEI R01 EY012223, P30 EY016665, Research to Prevent Blindness, CMP-NIH T32
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2539. doi:
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    • Get Citation

      Heather M Schmitt, Cassandra Schlamp, Huan Yang, Lian-Wang Guo, Robert W Nickells; Overexpression of histone deacetylase 3 (HDAC3) induces apoptosis in differentiated retinal neurons. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2539.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Histone deacetylase 3 (HDAC3) activity selectively induces apoptosis in differentiated neurons, but the mechanism for this selectivity is unknown. Importantly, HDAC3 has been found to play a major role in retinal ganglion cell (RGC) death after axonal injury. This study tests the hypothesis that overexpression of Hdac3 in mouse retinal neurons leads to cell-specific toxicity and that this phenomenon is dependent upon cell differentiation state.

Methods : Mouse 661W retinal precursor cells were nucleofected with Hdac3-mCherry, or HDAC3-FLAG, 24 hours prior to differentiation with 316nM staurosporine (STSN). All cells were then fixed at 0, 6, 12, and 24 hours post STSN addition to assess metrics of nuclear atrophy and apoptosis using immunofluorescence microscopy and live cell imaging. BAX activation was assessed by localization of a GFP-BAX fusion protein. Bax-dependency was evaluated on cells with mutated Bax using CRISPR/Cas9 and p53 dependency was evaluated in cells expressing a temperature sensitive mutant and a p53 promoter-Luciferase construct. Chromatin condensation was monitored using a mCherry-H2B fusion protein. C57BL/6 mice were given intravitreal injections of 1x109 units of AAV2-pgk-Hdac3mCherry virus 3, 4, and 5 weeks prior to assessing changes in RGC health.

Results : Hdac3 overexpression was achieved in 61.85% of 661Ws by 24 hours after nucleofection. Differentiated cells expressing Hdac3 exhibited significantly more activated BAX in comparison to undifferentiated cells (p=0.032), which was followed closely by chromatin condensation. Deletion of the Bax gene from these cells significantly reduced the level of Caspase-3 activation in response to HDAC3 expression (p=0.008). Both differentiated and undifferentiated groups exhibited altered p53-dependent gene expression. Experiments to test toxicity in response to overexpression of Hdac3 in-vivo are ongoing.

Conclusions : Hdac3 overexpression leads to BAX activation, chromatin condensation, and apoptosis only in differentiated cells. This process is likely Bax-dependent, but the requirement for p53-mediated gene expression remains unresolved. Further investigation is needed to identify the mechanism by which HDAC3 stimulates neuronal apoptosis both in-vitro and in-vivo.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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