The retina proteome

Figure 1. Schematic overview of the organization of the retina. Pigment epithelium cells are located nearest the choroid, while the nerve fiber layer is closest to the vitreous humor of the eye.

Retina transcriptome

Transcriptome analysis of the retina can be visualized with regard to the specificity and distribution of transcribed mRNA molecules (Figure 1). Specificity illustrates the number of genes with elevated or non-elevated expression in the retina compared to other tissues. Elevated expression includes three subcategory types of elevated expression:

• Tissue enriched: At least four-fold higher mRNA level in retina compared to any other tissues.
• Group enriched: At least four-fold higher average mRNA level in a group of 2-5 tissues compared to any other tissue.
• Tissue enhanced: At least four-fold higher mRNA level in retina compared to the average level in all other tissues.

Distribution, on the other hand, visualizes how many genes have, or do not have, detectable levels (nTPM≥1) of transcribed mRNA molecules in the retina compared to other tissues. As evident in Table 1, all genes elevated in retina are categorized as:

• Detected in single: Detected in a single tissue
• Detected in some: Detected in more than one but less than one-third of tissues
• Detected in many: Detected in at least a third but not all tissues
• Detected in all: Detected in all tissues

Figure 2. (A) The distribution of all genes across the five categories based on transcript specificity in retina as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (nTPM≥1) in retina as well as in all other tissues.

As shown in Figure 1, 744 genes show some level of elevated expression in the retina compared to other tissues. The three categories of genes with elevated expression in retina compared to other organs are shown in Table 1. In Table 2, the 12 genes with the highest enrichment in retina are defined.

Table 1. The number of genes in the subdivided categories of elevated expression in retina.

Table 2. The 12 genes with the highest level of enriched expression in retina. "Tissue distribution" describes the transcript detection (nTPM≥1) in retina as well as in all other tissues. "mRNA (tissue)" shows the transcript level in retina as nTPM values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in retina and the tissue with the second-highest expression level.

Protein expression of genes elevated in retina

In-depth analysis of the elevated genes in retina using antibody-based protein profiling allowed us to understand the distribution of the retina specific genes and their expression profiles. Proteins expressed in specific cell types or synapses were identified among the genes with elevated expression in the retina.

Proteins specifically expressed in pigment epithelial cells

Pigment epithelial cells aid the photoreceptor cells in various ways, including the supply of molecules essential for the conversion of light into nerve signals (phototransduction), that takes place in the photoreceptor cells. Retinaldehyde binding protein 1 (RLBP1) carries the molecules 11-cis-retinaldehyde and 11-cis-retinol and mutations in this gene have been shown to lead to different conditions causing vision loss, e.g. Cone-rod dystrophy. Beta-carotene oxygenase 2 (BCO2) is an enzyme involved in the biosynthesis of vitamin A, which is essential for phototransduction. Vitamin A is known to be transported to the retina from other parts of the body via blood circulation, however, the presence of BCO2 in pigment epithelial cells may indicate an additional site of vitamin A synthesis.

RLBP1
BCO2

Proteins specifically expressed in photoreceptor cells

An example of a protein located in the nuclei of photoreceptor cells is cone-rod homeobox (CRX), a transcription factor essential for normal photoreceptor function. There are two types of photoreceptor cells: rods and cones. In rods, phototransduction begins when light activates rhodopsin (RHO), followed by interaction with the rod-specific transducin. A component of the latter is G protein subunit gamma transducin (GNGT1). Another protein expressed in the outer segments is cyclic GMP-phosphodiesterase 6G (PDE6G). It converts cyclic GMP into 5' GMP in rods, a process known to lead to hyperpolarization and subsequent nerve signaling. S-antigen visual arrestin (SAG) turns off phototransduction by preventing interaction between rhodopsin and transducin. This allows activated rods to recover and consequently adapt to low light conditions.

CRX
RHO
GNGT1

PDE6G
SAG

Proteins involved in phototransduction in cones include g protein subunit gamma transducin 2 (GNGT2), guanylate cyclase activator 1B (GUCA1B) and arrestin 3 (ARR3). GNGT2 is a component of the cone-specific transducin, and like its rod-specific relative GNGT1, it is located in the outer segments. GUCA1B is a calcium-binding protein that activates photoreceptor guanylate cyclases. ARR3 is the cone-specific equivalent of SAG, and it is involved in the deactivation of cones.

GNGT2
GUCA1B
ARR3

Proteins specifically expressed in the outer limiting membrane

The outer limiting membrane is a row of cell junctions located where the inner and outer segments of photoreceptors connect. Crumbs 2 (CRB2) is a component of the Crumbs cell polarity complex, a protein important for many cellular processes during embryonic development. Mutation in this gene has been reported to cause congenital disorders such as retinitis pigmentosa where photoreceptor cells degrade and disappear. Transmembrane protein 221 (TMEM221) is another protein expressed in the outer limiting membrane. Its function is yet to be discovered.

CRB2
TMEM221

Proteins specifically expressed in the inner nuclear layer and ganglion cells

A transcription factor that may be involved in the development of cells in the inner nuclear layer (INL) is a visual system homeobox 2 (VSX2). VSX2 is expressed in the nuclei of a subset of cells in the INL. Recoverin (RCVRN), similarly to SAG, is known to block phototransduction in rods in darkness. Here we show that RCVRN is expressed in photoreceptor cells and in a subset of cells in INL. Calcium binding protein 5 (CABP5), is a member of a protein family involved in intracellular signaling. However, the exact function of this gene is unknown. Our analysis shows that CABP5 is expressed in the INL and ganglion cells in the human retina.

VSX2
RCVRN
CABP5

Proteins specifically expressed in the inner and outer plexiform layers

The outer and inner plexiform layers (OPL and IPL) contain synapses through which the nerve signals are transmitted between the different cells in the retina. Anoctamin 2 (ANO2) encodes a calcium-activated chloride channel expressed exclusively in the OPL. CPLX4 is a protein located in the IPL and may be involved in synaptic vesicle exocytosis. Glutamate metabotropic receptor 6 (GRM6) is, as the name suggests, one of several types of metabotropic receptors that bind the neurotransmitter L-glutamate in the central nervous system. In the retina, GRM6 is detected in both the OPL and IPL.

ANO2
CPLX4
GRM6

Gene expression shared between retina and other tissues

There are 233 group enriched genes expressed in the retina. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including retina, compared to all other tissues.

To illustrate the relation of retina to other tissue types, a network plot was generated, displaying the number of genes with a shared expression between different tissue types (Figure 3).

Retina shares most group enriched gene expression with the brain (n=176), which is expected based on that retina and brain share developmental origin and organization. GNG13 is a protein expressed exclusively in synapses in the retina and brain, as well as a subset of cells in the INL of the retina. It is a gamma subunit of a heterotrimeric G protein, which is known to be expressed in taste, retinal and neuronal tissues.

GNG13 - retina
GNG13 - cerebellum

Proteins analyzed in retina

Protein expression of selected genes with RNA expression elevated in retina or literature indicating function related to retina were analyzed in retina samples. The full list of genes used for protein profiling in the retina is found in Table 3.

Table 3. Following 113 genes have been analyzed in retina.

Retina function

The retina is a multilayered neural tissue that comprises the innermost layer of the eye, called the inner photosensitive layer. Similar to other parts of the central nervous system, it originates from the neuroepithelium, an outpocketing of the diencephalon. In the retina, light is converted into nerve signals that are transported to the visual centers in the brain by the optical nerve. As light enters the eye through the pupil and becomes refracted by the lens, it reaches the retina where it is absorbed. Photosensitive neuronal cells called photoreceptor cells transduce the light into nerve signals (this process is also known as phototransduction), which are then transmitted to different types of interneurons that modulate the nerve signaling before it is collected in the optical nerve.

There are two types of photoreceptor cells: rods and cones. Rod photoreceptors cells register the presence of light and allow vision during low light conditions while cones register red, green and blue colors and allow color vision. A layer of pigment epithelial cells provides nutrients, protection against UV radiation, and molecules essential for phototransduction to the photoreceptor cells. Four types of interneurons modulate nerve signaling by inhibiting and/or activating the cells they interact with, all located in the inner nuclear layer. Horizontal cells connect only to synapses of the photoreceptor cells, while bipolar cells transmit signals between photoreceptor cells and ganglion cells. Amacrine cells modulate signals coming from bipolar cells to ganglion cells. Ganglion cells are the last cells to receive and regulate molecular signals from the photoreceptor cells before nerve signals are collected in the optical nerve. They interact both with bipolar and amacrine cells. Muller glia cells, as the name suggests are glial cells. They maintain the structural integrity of the retina by stretching across all layers as well as buffer potassium and neurotransmitters.

Background

Here, the protein-coding genes expressed in retina are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in retina.

Transcript profiling was based on a combination of two transcriptomics datasets (HPA and GTEx), corresponding to a total of 14590 samples from 54 different human normal tissue types. The final consensus normalized expression (nTPM) value for each tissue type was used for the classification of all genes according to the tissue-specific expression into two different categories, based on specificity or distribution.

Relevant links and publications

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Fagerberg L et al., Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. (2014)
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600