Ablation of the Proapoptotic Genes Chop or Ask1 Does Not Prevent or Delay Loss of Visual Function in a P23H Transgenic Mouse Model of Retinitis Pigmentosa

<div><p>The P23H mutation in rhodopsin (Rho^P23H) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho^P23H rhodopsin transgene (<i>GHL⁺</i>). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for <i>GHL⁺</i>, <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i> animals between 4–28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of <i>Chop</i> and <i>Ask1</i> did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i>, a region that was severely degenerated in <i>GHL⁺</i> mice. Our results show that in the presence of the Rho^P23H transgene, the rate of decline in retinal sensitivity is similar in <i>Chop</i> or <i>Ask1</i> ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in <i>GHL⁺</i> mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.</p></div

ERG changes as a function of age in <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice.

<p>Maximal a- and b-wave amplitudes as a function of age in C57BL/6 (blue trace), <i>Chop^−/−</i> (orange trace) <i>GHL⁺</i> (black trace) and <i>GHL⁺/Chop^−/−</i> (red trace) mice. Colored lines are polynomial fits.</p

Age related changes in retinal function and morphology of <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> mice.

<p>(A) b/a wave ratio in C57BL/6, <i>Ask1^−/−</i>, <i>GHL⁺</i>, and <i>GHL⁺/Ask1^−/−</i> mice. (B) Representative sections of retinas from 16 week old C57BL/6, <i>Ask1^−/−</i>, <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> mice. <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> retinas had shortened OS (arrowheads). (C) Mean number of photoreceptor nuclei per column counted in the ONL. Error bars: ± SD. OPL: outer plexiform layer, ONL: outer nuclear layer, OS: outer segment. Scale bar 50 µm. (D) The variance in the number of photoreceptor nuclei per column counted in different zones of the retina in <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> mice, (<i>right panel</i>). Superior zone: 630 µm from the CMZ, central zone: 630 µm from optic nerve, and inferior zone: midpoint of inferior hemisphere.</p

b-wave threshold intensity (I_t) values.

<p>Threshold amplitude: 75 µV, I_t: Threshold intensity.</p

Histological analysis of 16 and 28 week old <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice.

<p>(A) Representative sections of retinas from 16 week old C57BL/6, <i>Chop^−/−</i>, <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice. <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> retinas had shortened OS (arrowheads). (B, <i>right panel</i>) Mean number of photoreceptor nuclei per column counted in the ONL at 16 weeks. The variance in the number of photoreceptor nuclei per column counted in different zones of the retina in (B, <i>left panel</i>) in 16 week. (C) Representative sections of retinas from 28 week old <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice. (D) The variance in the number of photoreceptor nuclei per column counted in different zones of the retina in 28 week old <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice. Superior zone: 630 µm from the CMZ, central zone: 630 µm from optic nerve, and inferior zone: midpoint of inferior hemisphere. Error bars: ± SD OPL: outer plexiform layer, ONL: outer nuclear layer, OS: outer segment. Scale bar 50 µm.</p

Rate of retinal degeneration in <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> mice.

<p>Maximal a- and b-wave amplitudes as a function of age in C57BL/6 (blue), <i>Ask1^−/−</i> (green) <i>GHL⁺</i> (black) and <i>GHL⁺/Ask1^−/−</i> (purple) mice. Error bars: ± SD. Colored lines are polynomial fits.</p

Histological analysis of 4 week old <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> retinas.

<p>(A) Representative sections of retinas from 4 week old C57BL/6, <i>Chop^−/−</i>, <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice (<i>n = 2–3</i>). (B) Mean number of photoreceptor nuclei per column counted in the ONL (<i>left panel</i>), and the variance in the number of photoreceptor nuclei per column counted in different zones of the retina in <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice, (<i>right panel</i>). Superior zone: 630 µm from the CMZ, central zone: 630 µm from optic nerve, and inferior zone: midpoint of inferior hemisphere. Error bars: ± SD OPL: outer plexiform layer, ONL: outer nuclear layer, OS: outer segment. Scale bar, 50 µm.</p

ERG responses in <i>GHL⁺</i> and <i>GHL⁺/Ask1^−/−</i> mice.

<p>Intensity-response functions of a- and b-waves in (A) 4, (B) 16, and (C) 28 week old C57BL/6 (blue), <i>Ask1^−/−</i> (green) <i>GHL⁺</i> (black) and <i>GHL⁺/Ask1^−/−</i> (purple) mice (<i>n = 7–14</i>). Error bars: ± SD.</p

Effect of <i>Chop</i> on the expression of phototransduction genes and visual function.

<p>(A). Quantitative RT-PCR analysis of 4 week old C57BL/6 and <i>Chop^−/−</i> mice (<i>n = 4</i>). Samples were normalized to β-actin levels. (B). Average scotopic ERG a- and b-wave waveforms from 4 week old C57BL/6 (blue trace) and <i>Chop^−/−</i> (orange trace) mice in response to 1 ms flashes (n = 7–9). (C) Intensity-response functions of a- and b-waves recorded from the same C57BL/6 and <i>Chop^−/−</i> mice. Horizontal lines and vertical intercepts on the abscissa (a-wave <i>I_t</i>: 50 µV, b-wave <i>I_t</i>: 75 µV) show the method used to determine threshold light intensities (<i>I_t</i>). (D) Age related changes in a- and b-wave threshold light intensities in C57BL/6 and <i>Chop^−/−</i> mice. Error bars are ± SD.</p

Age related changes in retinal function and sensitivity of <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice.

<p>(A) b/a wave ratio in C57BL/6, <i>Chop^−/−</i>, <i>GHL⁺</i>, and <i>GHL⁺/Chop^−/−</i> mice. (B and C) Age related changes in a- and b-wave threshold intensities in C57BL/6, <i>Chop^−/−</i>, <i>GHL⁺</i>, and <i>GHL⁺/Chop^−/−</i> mice (a- wave <i>I_t</i> = 50 µV, b- wave <i>I_t</i> = 75 µV). Error bars: ± SD.</p