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Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations

View Article: PubMed Central - PubMed

ABSTRACT

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

No MeSH data available.


PARP activity and PARylation in Pde6a mutant photoreceptors. The number of PARP activity-positive cells in the V685M, V685M*R562W, R562W, and D670G photoreceptors was strongly increased when compared with wt cells. The quantification of PARP activity-positive cells during the first 30 postnatal days (orange curve) identified peaks of activity at P12, P13, P15, and P21 in the V685M, V685M*R562W, R562W, and D670G mutants, respectively. Asterisks indicate significant levels of mutant versus wt PARP activity. A comparison with the number of dying, TUNEL-positive cells (red curve) in three out of four mutations showed a strong correlation with the peaks of PARP activity, while in the V685M*R562W model, PARP activity appeared to precede cell death (a–d). Similarly, a staining for the accumulation of PARylated proteins – products of PARP activity – showed a strong increase in all four Pde6a mutants when compared with wt cells. At the respective peaks of PARP activity, the number of PAR-positive cells was significantly increased in all four mutants (e–h). The images shown are representative for observations on at least six different specimens for each genotype. Values shown in line and bar graphs are mean±S.E.M., with n=6 in all cases. For easy reference, the peak time-points of PARP activity are stated in each such graph (a–d).
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fig1: PARP activity and PARylation in Pde6a mutant photoreceptors. The number of PARP activity-positive cells in the V685M, V685M*R562W, R562W, and D670G photoreceptors was strongly increased when compared with wt cells. The quantification of PARP activity-positive cells during the first 30 postnatal days (orange curve) identified peaks of activity at P12, P13, P15, and P21 in the V685M, V685M*R562W, R562W, and D670G mutants, respectively. Asterisks indicate significant levels of mutant versus wt PARP activity. A comparison with the number of dying, TUNEL-positive cells (red curve) in three out of four mutations showed a strong correlation with the peaks of PARP activity, while in the V685M*R562W model, PARP activity appeared to precede cell death (a–d). Similarly, a staining for the accumulation of PARylated proteins – products of PARP activity – showed a strong increase in all four Pde6a mutants when compared with wt cells. At the respective peaks of PARP activity, the number of PAR-positive cells was significantly increased in all four mutants (e–h). The images shown are representative for observations on at least six different specimens for each genotype. Values shown in line and bar graphs are mean±S.E.M., with n=6 in all cases. For easy reference, the peak time-points of PARP activity are stated in each such graph (a–d).

Mentions: Previous studies showed that PARP activity and the accumulation of its product poly(ADP-ribose) was increased during photoreceptor degeneration in the Pde6b mutant rd1 mouse retina.10 Hereafter, we refer to poly(ADP-ribose) as PAR and to the process as PARylation. We first performed an in situ PARP activity assay to analyse the temporal appearance of PARP activity in the photoreceptor layer in Pde6a mutants. The number of photoreceptor cells showing high PARP activity was quantified and peak activities were found at P12, P13, P15, and P21 for the V685M, V685M*R562W, R562W, and D670G mutants, respectively (Figures 1a–d).


Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations
PARP activity and PARylation in Pde6a mutant photoreceptors. The number of PARP activity-positive cells in the V685M, V685M*R562W, R562W, and D670G photoreceptors was strongly increased when compared with wt cells. The quantification of PARP activity-positive cells during the first 30 postnatal days (orange curve) identified peaks of activity at P12, P13, P15, and P21 in the V685M, V685M*R562W, R562W, and D670G mutants, respectively. Asterisks indicate significant levels of mutant versus wt PARP activity. A comparison with the number of dying, TUNEL-positive cells (red curve) in three out of four mutations showed a strong correlation with the peaks of PARP activity, while in the V685M*R562W model, PARP activity appeared to precede cell death (a–d). Similarly, a staining for the accumulation of PARylated proteins – products of PARP activity – showed a strong increase in all four Pde6a mutants when compared with wt cells. At the respective peaks of PARP activity, the number of PAR-positive cells was significantly increased in all four mutants (e–h). The images shown are representative for observations on at least six different specimens for each genotype. Values shown in line and bar graphs are mean±S.E.M., with n=6 in all cases. For easy reference, the peak time-points of PARP activity are stated in each such graph (a–d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig1: PARP activity and PARylation in Pde6a mutant photoreceptors. The number of PARP activity-positive cells in the V685M, V685M*R562W, R562W, and D670G photoreceptors was strongly increased when compared with wt cells. The quantification of PARP activity-positive cells during the first 30 postnatal days (orange curve) identified peaks of activity at P12, P13, P15, and P21 in the V685M, V685M*R562W, R562W, and D670G mutants, respectively. Asterisks indicate significant levels of mutant versus wt PARP activity. A comparison with the number of dying, TUNEL-positive cells (red curve) in three out of four mutations showed a strong correlation with the peaks of PARP activity, while in the V685M*R562W model, PARP activity appeared to precede cell death (a–d). Similarly, a staining for the accumulation of PARylated proteins – products of PARP activity – showed a strong increase in all four Pde6a mutants when compared with wt cells. At the respective peaks of PARP activity, the number of PAR-positive cells was significantly increased in all four mutants (e–h). The images shown are representative for observations on at least six different specimens for each genotype. Values shown in line and bar graphs are mean±S.E.M., with n=6 in all cases. For easy reference, the peak time-points of PARP activity are stated in each such graph (a–d).
Mentions: Previous studies showed that PARP activity and the accumulation of its product poly(ADP-ribose) was increased during photoreceptor degeneration in the Pde6b mutant rd1 mouse retina.10 Hereafter, we refer to poly(ADP-ribose) as PAR and to the process as PARylation. We first performed an in situ PARP activity assay to analyse the temporal appearance of PARP activity in the photoreceptor layer in Pde6a mutants. The number of photoreceptor cells showing high PARP activity was quantified and peak activities were found at P12, P13, P15, and P21 for the V685M, V685M*R562W, R562W, and D670G mutants, respectively (Figures 1a–d).

View Article: PubMed Central - PubMed

ABSTRACT

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

No MeSH data available.