Inhibitory peptide of mitochondrial μ-calpain protects against photoreceptor degeneration in rhodopsin transgenic S334ter and P23H rats

Abstract

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.

Figure 1. Determinations of photoreceptor cell death in S334ter rat retinas.

A) TUNEL assay of retinal sections of S334ter rats. Eyes were enucleated at PN 13, 14, 15, 18, 21, 24, 27, or 30 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 2. Effects of intravitreal injection of Tat-µCL on photoreceptor cell death in S334ter rats.

A) TUNEL staining of retinal sections of S334ter rats treated with Tat-µCL. S334ter rats received intravitreal injection of 2 µl of vehicle (PBS), 4 mM PD150606, or 20 mM Tat-µCL at PN 15 days. Eyes were enucleated at PN 18 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL at PN 18 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). ***P<0.001 versus vehicle (t-test). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 3. Effects of eye-drop applications of Tat-µCL on photoreceptor cell death in S334ter rats.

A) TUNEL staining of retinal sections of S334ter rats treated with eye-drops containing Tat-µCL. Eye-drops containing vehicle (PBS) or 20 mM Tat-µCL were administered from PN 13 to 17 days. Eyes were enucleated at PN 18 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL at PN 18 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). ***P<0.001 versus vehicle (t-test). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 4. Effects of an intravitreal injection or eye drop applications of Tat-µCL on ERG in S334ter rats.

S334ter rats received an intravitreal injection of 2 µl of 20 mM Tat-µCL at PN 15 days (▪). Another group of S334ter rats received eye-drops containing 20 mM Tat-µCL from PN 13 to 55 days (•). Scotopic ERGs were recorded at PN 18, 21, 24, 28, 35, 42, 49, and 56 days. A) Mean amplitudes of photoreceptor-derived a-waves. B) Mean amplitudes of Müller cells-derived b-waves. Data are expressed as means ± standard deviation (n = 8 eyes (8 rats) per group). *P<0.05 and **P<0.01 versus the none-treated group (○) (t-test).

Figure 5. Effects of eye drop applications of Tat-µCL on photoreceptor cell death in P23H rats.

A) TUNEL of retinal sections of P23H rats treated with eye-drops containing Tat-µCL. Eye-drops containing saline (PBS), 1 mM Tat-µCL in saline, or 1 mM Tat-µCL in 0.1% HA were administered from PN 14 to 49 days. Eyes were enucleated at PN 30, 40, or 50 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL at PN 30, 40, and 50 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). *P<0.05 and **P<0.01 versus the saline-treated group (t-test). Abbreviations: ONL, outer nuclear layer.

Figure 6. Determination of nuclear translocation of AIF in P23H rat retinas.

A) Eyes were enucleated at PN 40 days, and retinal sections were stained with AIF (red), TUNEL (green) and DAPI (blue). AIF was detected in photoreceptor cell nuclei. Arrows indicate localization of AIF in TUNEL-positive photoreceptor nuclei. B) Effects of eye-drop applications of Tat-µCL on nuclear translocation of AIF in P23H rats. Eye-drops containing saline (PBS), 1 mM Tat-µCL in saline, or 1 mM Tat-µCL in 0.1% HA were administered from PN 14 to 39 days. Eyes were enucleated at PN 40 days. Retinal sections were stained with AIF (red) and DAPI (blue). White circles indicate translocation of AIF inside photoreceptor nuclei (shown by pink color). Abbreviations: OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 7. Effects of eye-drop applications of Tat-µCL on thickness of retinal layers in P23H rats.

A) Eye-drops containing saline (PBS), 1 mM Tat-µCL in saline, or 1 mM Tat-µCL in 0.1% HA were administered from PN 14 to 89 days. Eyes were enucleated at PN 30, 70, or 90 days. Retinal sections were stained with hematoxylin and eosin. B) Quantitative analysis of the thickness of ONL at PN 30, 70, and 90 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). *P<0.05, **P<0.01, and ***P<0.001 versus the saline-treated group (t-test). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 8. Effects of eye-drop applications of Tat-µCL on ERG in P23H rats.

A) Representative ERG traces. Eye-drops containing vehicle (PBS) or 1 mM Tat-µCL in saline were administered to P23H rats from PN 14 to 89 days. Scotopic ERGs were recorded at PN 30, 70, or 90 days. B) Mean amplitudes of photoreceptor-derived a-waves. C) Mean amplitudes of Müller cells-derived b-waves. Data are expressed as means ± standard deviation (n = 8 eyes (8 rats) per group). *P<0.05 and **P<0.01 versus the vehicle-treated group (t-test).