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By Shannon R. Barwick, Haiyan Xiao, David Wolff, Jing Wang, Elizabeth Perry, Brendan Marshall, and Sylvia B. Smith

Extract from the article published in Experimental Eye Research, Volume 230, May 2023, 109462, ISSN 0014-4835, DOI: https://doi.org/10.1016/j.exer.2023.109462

Editor’s Highlights

• The slowly-progressing Rho^P23H/+ mouse, a model of autosomal dominant Retinitis pigmentosa, was treated with a high-specificity sigma 1 receptor agonist ligand 3x/week at 0.5 mg/kg.
• Taken collectively, the in vivo and in vitro data provide the first evidence that targeting Sig1R can rescue visual function and structure in the Rho^P23H/+ mouse model.

Abstract

Retinitis pigmentosa (RP) is a group of devastating inherited retinal diseases that leads to visual impairment and oftentimes complete blindness. Currently no cure exists for RP thus research into prolonging vision is imperative. Sigma 1 receptor (Sig1R) is a promising small molecule target that has neuroprotective benefits in retinas of rapidly-degenerating mouse models. It is not clear whether Sig1R activation can provide similar neuroprotective benefits in more slowly-progressing RP models. Here, we examined Sig1R-mediated effects in the slowly-progressing Rho^P23H/+ mouse, a model of autosomal dominant RP. We characterized the retinal degeneration of the Rho^P23H/+ mouse over a 10 month period using three in vivo methods: Optomotor Response (OMR), Electroretinogram (ERG), and Spectral Domain-Optical Coherence Tomography (SD-OCT). A slow retinal degeneration was observed in both male and female Rho^P23H/+ mice when compared to wild type. The OMR, which reflects visual acuity, showed a gradual decline through 10 months. Interestingly, female mice had more reduction in visual acuity than males. ERG assessment showed a gradual decline in scotopic and photopic responses in Rho^P23H/+ mice. To investigate the neuroprotective benefits of Sig1R activation in the Rho^P23H/+ mouse model, mutant mice were treated with a high-specificity Sig1R ligand (+)-pentazocine ((+)-PTZ) 3x/week at 0.5 mg/kg and examined using OMR, ERG, SD-OCT. A significant retention of visual function was observed in males and females at 10 months of age, with treated females retaining ∼50% greater visual acuity than non-treated mutant females. ERG revealed significant retention of scotopic and photopic b-wave amplitudes at 6 months in male and female Rho^P23H/+ mice treated with (+)-PTZ. Further, in vivo analysis by SD-OCT revealed a significant retention of outer nuclear layer(ONL) thickness in male and female treated Rho^P23H/+ mice. Histological studies showed significant retention of IS/OS length (∼50%), ONL thickness, and number of rows of photoreceptor cell nuclei at 6 months in (+)-PTZ-treated mutant mice. Interestingly, electron microscopy revealed preservation of OS discs in (+)-PTZ treated mutant mice compared to non-treated. Taken collectively, the in vivo and in vitro data provide the first evidence that targeting Sig1R can rescue visual function and structure in the Rho^P23H/+mouse. These results are promising and provide a framework for future studies to investigate Sig1R as a potential therapeutic target in retinal degenerative disease.

Introduction

Retinitis pigmentosa (RP), a group of devastating inherited retinal degenerative diseases, leads to death of rod photoreceptor cells (rod/cone dystrophy) or the death of cone photoreceptor cells (cone/rod dystrophy) (Gargini et al., 2007). RP can be inherited as autosomal dominant (adRP), autosomal recessive (arRP), or X-linked. The majority of RP cases are autosomal recessive; adRP cases comprise the second largest number of cases with most mutations occurring in the RHO gene and are non-syndromic RP (Mendes et al., 2005). RP typically leads to the loss of rod photoreceptor cells initially with subsequent loss of cones (Hamel, 2006). It presents most often in early adulthood and patients complain of night blindness and decreased peripheral vision. The progression of RP is typically slow but due to the heterogeneous nature of the disease it is difficult to predict (van Soest et al., 1999). Currently, no cure exists for RP and therapies focus on stem cell therapy (Carr et al., 2013; Zakrzewski et al., 2019), gene therapy (Latella et al., 2016; Vandenberghe et al., 2011: Yu et al., 2017) and retinal implants (Hallum and Dakin, 2021). While these are promising approaches, therapeutic targets are a propitious option that can also benefit a wide range of RP patients with varying disease origins.

Sigma 1 receptor (Sig1R) is a unique protein with robust neuroprotective retinal properties (Smith et al., 2018). It is a small (∼27kD) transmembrane protein with no known mammalian homologs. Sig1R is a pluripotent modulator of cell survival (Su et al., 2016) and has been classified as a novel target for treatment of neurodegenerative diseases (Nguyen et al., 2015). It is abundantly expressed throughout the eye including all retinal cell types, notably photoreceptors (Ola et al., 2001; Wang et al., 2002).

The first report addressing whether Sig1R modulates photoreceptor cell survival in vivowas from Hara’s group. They administered SA4503, a Sig1R ligand, intravitreally in wild type mice 1 h prior to inducing photoreceptor cell death with intense light exposure. They reported improved dark-adapted ERGs and decreased photoreceptor cell loss in SA4503-treated v. non-treated mice (Shimazawa et al., 2015). Sig1R-mediated retinal protection has been investigated in genetic models of photoreceptor cell degeneration. Our lab utilized the Pde6b^rd10/J (rd10) mouse model to test activation of Sig1R (Wang et al., 2016). The rd10 mouse has a mutation in the beta subunit of the cGMP-phosphodiesterase gene (Chang et al., 2002, 2007) and develops a rapid photoreceptor cell degeneration. Upon treatment with a high affinity Sig1R ligand (+)-pentazocine ((+)-PTZ), mice showed improved photopic ERGs and histological assessment revealed cone preservation (Wang et al., 2016). To confirm that cone photoreceptor cell protection was directly related to Sig1R activation, our lab generated rd10 mice lacking Sig1R (rd10/Sig1R^−/−). These mice were treated with/without (+)-PTZ and showed no improvement in photopic ERG and no cone preservation suggesting Sig1R is required for (+)-PTZ-mediated cone rescue (Wang et al., 2016). The Guo lab also investigated lack of Sig1R in rd10 mice (reared in dim light to reduce rapid photoreceptor cell loss) and found that rd10/Sig1R^−/− mice had a retinal phenotype that was worse than rd10/Sig1R^+/+ mice (Yang et al., 2017).

The rd10 mouse provided compelling evidence that activation of Sig1R is potentially therapeutic though the degenerative process is quite rapid. The current study explored the role of Sig1R-mediated retinal neuroprotection in a model with a more slowly progressing disease that more faithfully recapitulates RP in humans, the Rho-P23H mouse model. The P23H mutation of rhodopsin accounts for ∼10% of all cases of adRP (Chen et al., 2014).

A number of laboratories have attempted to create an adRP model with the P23H mutation of rhodopsin (Barwick et al., in press), and the one established by the Palczewski laboratory authentically recapitulates the human disease. A cohort of 19 human adRP patients with the RHO-P23H mutation were evaluated in conjunction with the knock-in Rho-P23H mouse model that was generated. The mouse demonstrates a slowly progressing loss of ERG function over many months, similar to patients carrying the Rho^P23H/+ mutation, and a slow progressive retinal degeneration in which gradual shortening and disorganization of outer segments precedes rod photoreceptor cell death. A notable feature of the Rho^P23H/+ mouse model is that its degeneration pattern shows regional retinal degenerative differences such that the ventral (inferior) retina degenerates more rapidly than the dorsal (superior) retina, which is similar to the inferior-superior differences observed in human RP patients (Sakami et al., 2011). In aggregate, the aforementioned similarities of the humanized mouse model created in the Palczewski laboratory to human adRP patients (Dias et al., 2018) make this model a particularly attractive tool for investigating mechanisms of disease and interventional strategies.

The present work investigated Sig1R activation in this model. Earlier reports of the Rho^P23H/+ mouse reported ERG responses in a small number of mice, however no other functional tests were performed. Here, we conducted comprehensive functional and morphological assessments of the retinal degeneration over a 10 month period and evaluated consequences of Sig1R activation in the Rho^P23H/+ model.