Abstract
Poly-ADP-ribose-polymerase (PARP) relates to a family of enzymes that can detect DNA breaks and initiate DNA repair. While this activity is generally seen as promoting cell survival, PARP enzymes are also known to be involved in cell death in numerous pathologies, including in inherited retinal degeneration. This ambiguous role of PARP makes it attractive to have a simple and fast enzyme activity assay, that allows resolving its enzymatic activity in situ, in individual cells, within complex tissues. A previously published two-step PARP activity assay uses biotinylated NAD+ and streptavidin labelling for this purpose. Here, we used the fluorescent NAD+ analogues ε-NAD+ and 6-Fluo-10-NAD+ to assess PARP activity directly on unfixed tissue sections obtained from wild-type and retinal degeneration-1 (rd1) mutant retina. In standard UV microscopy ε-NAD+ incubation did not reveal PARP specific signal. In contrast, 6-Fluo-10-NAD+ resulted in reliable detection of in situ PARP activity in rd1 retina, especially in the degenerating photoreceptor cells. When the 6-Fluo-10-NAD+ based PARP activity assay was performed in the presence of the PARP specific inhibitor olaparib, the activity signal was completely abolished, attesting to the specificity of the assay. The incubation of live organotypic retinal explant cultures with 6-Fluo-10-NAD+, did not produce PARP specific signal, indicating that the fluorescent marker may not be sufficiently membrane-permeable to label living cells. In summary, we present a new, rapid, and simple to use fluorescence assay for the cellular resolution of PARP activity on unfixed tissue, for instance in complex neuronal tissues such as the retina.
Highlights
(ADP-ribose) polymerase (PARP) is a family of enzymes involved in numerous cellular processes [1, 2]
The current assay to evaluate Poly (ADP-ribose) polymerase (PARP) activity ex vivo on tissue sections is using biotinylated NAD+, which in a second step is recognized by fluorescently labelled streptavidin [12]
When this two-step assay was performed on rd1 P11 retinal sections, a large number of positive cells was seen in the outer nuclear layer (ONL), while essentially no positive cells were seen in the wild type (WT) condition at the same age (Fig 2A)
Summary
(ADP-ribose) polymerase (PARP) is a family of enzymes involved in numerous cellular processes [1, 2]. An important function of PARP is to detect DNA damage and to activate the enzymatic machinery involved in the DNA repair process. To this effect, PARP uses NAD+ as a substrate to build poly-ADP ribose polymers (PAR) [3]. A specific type of caspase-independent programmed cell death, called PARthanatos, appears to be triggered by the accumulation of PAR polymers [5, 6].
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