Point mutation KI mouse

Case Study

Model with heterozygous point mutations, exactly homologous to human retinitis pigmentosa (RP).

‍Sothilingam V, et al. Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype. Hum Mol Genet. 2015.

Mutations in the PDE6A gene can cause rod photoreceptor degeneration and the blinding disease retinitis pigmentosa.

Model: Novel mouse model for the Pde6a^R562W point mutation in combination with an existing line carrying the V685Mpoint mutation to generate compound heterozygous Pde6a^V685M/R562W animals, exactly homologous to a case of human RP.

Aim: Predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window of opportunity for clinical interventions.

Results: The study provides a rational basis for predictions on human RP phenotypes and disease progression in compound heterozygous situations, and suggests the targeting of non-apoptotic processes as a feasible treatment approach.

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Figure 1. Loss of PDE6A expression causes cGMP accumulation.

Excessive accumulation of cGMP and subsequent rod photoreceptor death, followed by a mutation-independent, secondary death of cone photoreceptors.

A, B) In the PN11 wildtype (wt) retina (A), immunostaining for PDE6A shows strong protein expression in photoreceptor OS.

In contrast, at the same age, in the compound heterozygous V685M/R562W retina (C), the protein is essentially absent.

C, D) At PN11, wt retina is essentially negative for cGMP immunoreactivity (C).

PDE6A mutants, however, display individual rod photoreceptor cells that have accumulated large amounts of cGMP (D).

E) The quantification of cGMP-positive cells in the ONL and the PDE6A pixel intensity in the OS (arbitrary units; AU) shows an inverse correlation. Images are representative for immunostaining performed on retinal sections from at least three independent animals for each genotype.

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Figure 2. Photoreceptor cell death and survival.

A-C) The TUNEL assay in the wt retina occasionally labeled cells dying due to developmental processes.

D-F) In Pde6a mutants, photoreceptor cell death was dramatically increased.

The images show the situation at P12, P15 and P21, time-points corresponding to the peak of cell death..

G, H) The line graph at the bottom left (G) illustrates the progression of photoreceptor cell death as evidenced by the TUNEL assay in different Pde6a mutants. The green line corresponds to the V685M*R562W mutant.

The peak times as well as the peak amplitudes correspond to the speed of retinal degeneration, which is illustrated by the loss of photoreceptors (H).

Images are representative for TUNEL assays performed on retinal sections from at least three independent animals; quantifications in G, H include data from 3-7 animals per genotype and time-point.

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Figure 3. Photoreceptor degeneration in Pde6a mutants correlates with calpain, not caspase, activity.

Calpain activity is often associated with non-apoptotic forms of cell death, and in all Pde6a mutants it is strongly correlated in time with the progression of photoreceptor cell death.

A) Quantification of caspase-3-positive cells, which shows only extremely low numbers of cells at the respective peaks of degeneration, with no significant (n.s.) differences to wt.

B) The progression of calpain activity was analyzed over time and showed a strong correlation to the extent of cell death and the progression of retinal degeneration.