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M. V. Alavi, F. Paquet-Durand, N. Fuhrmann, P. Ekström; A Histone Deacetylase Activity Assay Enables the Analysis of Intracellular HDAC Activity of Single Cells in the Retina of the Rd1-Mouse. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4471.
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© ARVO (1962-2015); The Authors (2016-present)
Histone acetylation/deacetylation alters the chromatin structure, activating or repressing transcription. It plays a fundamental role in cell proliferation, cell differentiation and cell death. The deacetylation step is performed by the Trichostatin A (TSA) sensitive histone deacetylases Class I and II (HDACs) and the TSA insensitive Class III HDACs (Sirtuins). Since the cell death of photoreceptors leads to various retinal degenerations, our aim was to establish an HDAC-assay that could be performed on retinal sections, thus assessing the various HDAC-activities in defined cells from either normal or degenerating retinas.
Retinas from mice with inherited retinal photoreceptor degeneration (rd1 mouse) or from wild-type counterparts were cryosectioned and assessed for deacetylation activity. The latter used a fluorescence based technique, that enabled us to use fluorescence microscopy to monitor the deacetylation of a given substrate over time.
The Km-values for the general HDAC activity as well as the Sirtuin activity in the whole retina were determined. They were in the same range as described. Furthermore, we could show that the total HDAC activity was the sum of the activity of the Class I/II HDACs and that of the Sirtuins. Although the intracellular HDAC activity was monitored indirectly, in the sense that the deacetylated substrate carrying the read-out signal diffused out of/away from the cells after the deacetylation had taken place, we consider our results as proof-of-principle for the technique. Importantly, with extended incubation time and an additional fixation step, we observed that several cells in the photoreceptor layer of the rd1-mouse retina showed a strong HDAC-activity that was absent in the wild-type controls. This HDAC-activity could be assigned to an increased activity of the Class I/II HDACs, because it could be suppressed by Trichostatin A, whereas nicotinamide, a Sirtuin inhibitor, was ineffective in this respect.
HDAC activity could be measured in situ in retinal sections and a correlation between Class I/II HDAC acitivity and retinal degeneration was observed. These results open up a new field of research in retinal degeneration.
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