Retina pigmentosa

Retina pigmentosa

Retina pigmentosa is a retinal degenerative disease characterized by night blindness, loss of periphery vision, and potentially resulting in complete loss of vision. RP is the most common inherited retinal dystrophy affecting approximately 100,000 people in the United States and one in every 2000 individuals worldwide (Phillips, Otteson & Sherry, 2010). The condition mainly affects the rod photoreceptors and retinal pigment epithelium (Farkas, Grant & Pierce, 2012). Retinitis pigmentosa (RP) is a genetically heterogeneous disorder and can be caused by defects in as many as 100 genes. Affected individuals can inherit the condition as an autosomal dominant, autosomal recessive, autosomal X-linked trait, or in rare cases, mitochondrial or digenic forms (Phillips, Otteson & Sherry, 2010).

A common characteristic of all forms for RP is the loss of night and peripheral vision, followed by a progressive loss of central vision. Although a majority of mutated gene expression occurs exclusively in rods photoreceptors, typically, secondary degeneration of cone receptors follows the degeneration of rod receptors (Veleri et al., 2015). Due to limited treatment options, progressive vision loss is the prognosis for most patients. Despite limited understanding of the molecular underpinning of initiation and progression of retina pigmentosa, the development of animal models of the condition, alongside high-throughput RNA-sequencing provides an opportunity to study the underlying cellular and molecular changes in the condition. This paper examines RNA-Seq of mouse models rd1 (associated with early-onset, severe retinal degeneration) and rd10 (associated with early-onset mild retinal degeneration) in exploring human retinal degeneration pathogenesis.

Overview of Retina Structure and Function

Light is essential for daily functioning and behavior in most organisms. In vertebrates, photoreceptors in the retina capture light, and the brain receives their output to reconstitute images. Sight is fundamental for the quality of life, and vision impairment is highly incapacitating (Veleri et al., 2015). The cause of visual loss dysfunction is either the blockage of light from reaching the neural retina or the inability of the retina to detect or transmit light-triggered signals to the brain. It is the inability of the retina to detect or transmit signals that characterize retinal degenerative diseases.

The unique structure of the retina enables the perception, integration, and transmission of visual information. Synaptic layers separate the three cellular layers consisting of six major types of neurons. Photoreceptors are the light-sensitive cells in the retinae of two subtypes: rods and cones (Veleri et al., 2015). Rods photoreceptors are responsible for dim light regions while cone photoreceptors mediate color vision and high visual acuity under bright light conditions. In humans and most other mammals, rods significantly outnumber cones, for example, the human retina contains 105million rods and 6 million cones (Veleri et al., 2015). The retinal pigment epithelium (RPE) is an additional cell layer that underlies the retina and barricades the photoreceptors and the choroidal blood supply. The RPE is vital in the functioning of photoreceptors as it facilitates a two-way transfer of nutrients and waste products and retinal recycling.

While cones photoreceptors in the human retina are classified into long, medium, and short-wavelength cones, there is only one type of rod photoreceptor in humans, mice, and other vertebrates. Photoreceptors need to function properly for vision. Mutations that affect photoreceptor function or survival, thereby disrupting the phototransduction process lead to vision loss (Weber & Langman, 2019). Additionally, defects in the RPE and other retinal cell types can also result in photoreceptor dysfunction and retinal degeneration.

Overview of RNA-Seq

Dysfunction or death of photoreceptor cells is a significant cause of incurable vision loss. Advances made in the last two decades in discovering genes and genetic defects causing retinal diseases has shifted the focus to uncovering disease mechanisms and formulation of treatments, particularly following the successful application of gene therapy in some congenital blindness in humans (Veleri et al., 2015). Mouse mutants, both spontaneous and laboratory-generated, valuably provide fundamental insights into the normal development of the retina and for deciphering disease pathology. RNA-Seq is the most robust transcriptional model used today by researchers.

RNA-Seq is a highly effective NGS method that allows a detailed study of transcriptomes. It provides a high degree of accuracy and unprecedented resolution of gene expression, which enables the study of RNA editing, isoform quantification, and alternative splicing (Farkas et al., 2015). RNA-Seq has fast become the preferred method for studying transcriptomes of tissues and individual cells. RNA’s non-dependency on annotated transcriptome and its extreme high-throughput lays the foundation for novel genetic discovery, making it advantageous over previous methods such as microarrays. Besides, RNA-derived transcriptomes offer a platform for a wide scope of research aimed at identifying potential therapeutic targets in the treatment of retinal pigmentosa (Dias et al., 2018).

Despite the rapid evolution of the protocols for the generation of cDNA libraries from total RNA, the underlying principles remain the same: isolating and fragmenting high-quality mRNA to a user-defined length (Farkas et al., 2015). A conversion of fragmented mRNA to double-stranded cDNA and the litigation of common adapters to the end of the cDNA follow. After sequencing, the data is aligned to the genome or transcriptome ready for analysis in gene expression, alternative or aberrant splicing, expression of individual exons, polymorphisms, and insertions and deletions.

Retinal Degeneration 1 (Pde6brd1) and 10 (Pde6brd10)

The use of animal models facilitates the elucidation of cellular mechanisms underlying human disease. Mice are the most widely used models of human disease. They are easy to manage in a laboratory setting, and several mutants of retinal disease already exist or can be generated easily for investigations (Veleri et al., 2015). Pde6brd1 (rd1) and Pde6brd10 (rd10) mouse models of RP are caused by recessive mutations in β-phosphodiesterase, a gene encoding that is rod-specific.

rd1, originally known as rodless mouse, is the first retinal degeneration, and the mutation has been found in commonly laboratory inbred strains and wild-inbred strains (Phillips, Otteson & Sherry, 2010). Mice homozygous for the rd1 mutation experience an early-onset severe retinal degeneration caused by a murine viral insert and a second mutation in exon 7 of the Pde6b gene encoding the beta subunit of cGMP-PDE (Chang et al., 2002). Human patients suffering from autosomal recessive retinitis pigmentosa also have mutations in the gene encoding the beta subunit of cGMP-PDE, a condition mirroring that caused by mouse Pde6brd1 mutation.

rd10 is the fifteenth retinal degeneration. Mice homozygous for the rd10 mutation exhibit retinal degeneration with sclerotic retinal vessels at 4 weeks of age. Genetic analysis shows that the condition is due to an autosomal recessive mutation that maps to mouse Chr5 (Chang et al., 2002). Sequence analysis shows that a missense mutation in exon 13 of the beta subunit of the rod phosphodiesterase gene causes retinal degeneration. The exon 13 missense mutation in mice may provide a good model for studying the pathogenesis of autosomal recessive retinitis pigmentosa in humans. Its later onset and milder retinal degeneration potentially make it better than rd1 for experimental pharmaceutical-based therapy for retinitis pigmentosa.

RNA-SEq of Retinal Transcriptome Analysis

It involved a review of literature on RNA-SEq of retinal transcriptome analysis of the rd1 and r10 murine model of retinal degeneration. Uren et al. (2014 investigated transcriptional changes in the rd10 mouse model of RP using RNA-Seq retinal transcriptome analysis. Results from their study showed that retinas from rd10 mice exhibit loss of high-expression vision and RP related genes, increased relative expression of immune response genes, variations in transcriptional and post-transcriptional control, elevated expression of Muller-specific genes following a decrease in rod-specific genes, and degenerated data exhibit changes in splicing. The results for rd10 show the loss of rod-specific transcripts and the increased relative expression of Muller-specific transcripts, suggesting the importance of reactive gliosis and innate immune activation in retinal pigmentosa (Uren et al., 2014). Also, there were significant changes in relative isoform usage among neuronal differentiation and morphogenesis genes, which includes a marked shift to shorter transcripts.

Xu et al. (2019) studied the effect of intravitreally injected metformin on retinal degeneration in rd1. In the study, they used RNA-Seq and bioinformatic analysis in the “Met” treatment group and control group. High-throughput gene sequencing analysis obtained the genes with significant differences between the experimental group and the comparison group. The results showed that metformin delays visual impairment and rescues photoreceptors from apoptosis in rd1 mice (Xu et al., 2019). Also, there are changes in the gene expression profile of rd1 mice after metformin treatment.

The results show that intravitreally injected metformin produces a protective effect on the visual function of rd1 mice, suggesting that metformin can delay but not entirely prevent the progression of RP in rd1 mice.

Discussion

RNA-Seq analysis of rd10 indicates inner retina remodeling and possible Muller dedifferentiation, while that of rd1 suggests that metformin is a potential treatment for RP (Uren et al., 2014, Xu et al., 2019). rd1 mice carry mutations in the beta subunit of rod-cGMP of PDE6β, which results in the accumulation of cGMP in the retina. The accumulation activates cGMP dependent protein kinase (PKG), which plays a significant role in photoreceptor degeneration and early-onset severe retinal degeneration in rd1 mice. The study showed that metformin produced antiapoptotic effects in photoreceptors, which delayed functional impairment in rd1 mice (Xu et al., 2019).

Currently, no effective treatments for RP exist. The integrity of second- and third-order retinal neurons, together with their ability in processing and transmitting visual signals to the brain, determines success in restoring vision. But evidence shows that the death of photoreceptors leads to secondary remodeling of the remaining neurons in the retina (Phillips, Otteson & Sherry, 2010). The remodeling involves various negative plastic changes, including loss of glutamate receptors, loss or sprouting of neuronal processes, reactive gliosis, and cellular migration. Researchers have reported retinal remodeling in many animal models, including rd1 and rd10 mice (Phillips, Otteson & Sherry, 2010).

The significant temporal overlap between degeneration and normal cellular and synaptic development resulting from the early onset makes it difficult to identify specific origins of retinal remodeling in rd1 (Phillips, Otteson & Sherry, 2010). On the contrary, analysis of degeneration and retinal remodeling in the context of a developed and functional retinal is made possible by the delayed onset and slower rate of progression of degeneration in rd10. As a result, the rd10 mouse is finding its uses in developing new experimental therapies of RP. Therefore, developing successful treatments for RP and evaluating their effectiveness, and understanding of the structural, neurochemical, and functional consequences of remodeling is vital.

Conclusion

Retina pigmentosa is a retinal degenerative disease characterized by night blindness, loss of periphery vision, which potentially leads to complete loss of vision. The condition mainly affects the rod photoreceptors. The use of animal models facilitates the elucidation of cellular mechanisms underlying human disease. Mice are the most widely used models of human disease. RNA-Seq is a highly effective NGS method that allows a detailed study of transcriptomes. It provides a high degree of accuracy and unprecedented resolution of gene expression, which enables the study of RNA editing, isoform quantification, and alternative splicing. RNA-Seq analysis of rd10 indicates inner retina remodeling and possible Muller dedifferentiation, while that of rd1 suggests that metformin is a potential treatment for RP. Therefore, for developing successful therapies for RP and evaluating their effectiveness, and understanding of the structural, neurochemical, and functional consequences of remodeling is vital.