The retina proteome

The retina is a multilayered neural tissue located in the eye and converts light into nerve signals that are transported to the visual centers in the brain by the optical nerve. The retina originates from neuroepithelium (an outpocketing of the diencephalon) and consists of a network of neuronal and glial cells connected via synapses. Consequently, it shares the developmental origin and organization with other parts of the central nervous system (CNS). Transcriptome analysis shows that 79% (n=15630) of all human proteins (n=19670) are expressed in the retina and 310 of these genes show an elevated expression in the retina compared to other tissue types.

  • 310 elevated genes
  • 87 enriched genes
  • 79 group enriched genes
  • Retina has most group enriched gene expression in common with brain


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


Retina transcriptome

Transcriptome analysis of the retina can be visualized with regard to 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 that have, or do not have, detectable levels (NX≥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

A. Specificity

B. Distribution

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 (NX≥1) in retina as well as in all other tissues.

As shown in Figure 1, 310 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. Number of genes in the subdivided categories of elevated expression in retina.

Distribution in the 37 tissues
Detected in singleDetected in someDetected in manyDetected in all Total
Specificity
Tissue enriched 061233 87
Group enriched 048301 79
Tissue enhanced 0341019 144
Total 014315413 310

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

Gene Description Tissue distribution mRNA (tissue) Tissue specificity score
RHO rhodopsin Detected in some 415.0 151
OPTC opticin Detected in some 245.8 92
RCVRN recoverin Detected in some 208.6 75
RBP3 retinol binding protein 3 Detected in some 135.2 45
CRX cone-rod homeobox Detected in some 125.2 42
GNAT1 G protein subunit alpha transducin 1 Detected in some 171.9 39
AIPL1 aryl hydrocarbon receptor interacting protein like 1 Detected in some 123.7 37
AC010319.2 Detected in some 41.6 32
TSPAN10 tetraspanin 10 Detected in some 117.4 29
SAG S-antigen visual arrestin Detected in some 215.8 26
NR2E3 nuclear receptor subfamily 2 group E member 3 Detected in some 50.2 26
RPE65 RPE65, retinoid isomerohydrolase Detected in some 268.3 25


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 in synapses were identified among the genes with elevated expression in the retina.

Proteins specifically expressed in pigment epithelial cells

Pigment epitheli cells produce melanin to protect photoreceptor cells from UV-radiation. Our analysis revealed that, in addition to melanocytes in the skin, melan-A (MLANA) is also expressed in pigment epitheli cells. Pigment epitheli cells are also involved in the transport of nutrients and ions between photoreceptor cells and blood vessels in the choroid. Another protein identified in pigment epitheli cells is bestrophin 1 (BEST1); it is known to form a bicarbonate transporting channel. Solute carrier family 16 member 8 (SLC16A8) plays a role in the transport of molecules and here we show that it is located in the cell membrane of pigment epitheli cells.


MLANA

BEST1

SLC16A8

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. The process of conversion of light to nerve signals, also called phototransduction, involves interaction between numerous proteins in the outer segments of photoreceptor cells,. 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, as illustrated by integrin subunit beta 8 (ITGB8), which is a component of the integrin complex. Integrin complexes are important for cell to cell and cell to matrix interactions. Crumbs 2 (CRB2) is another protein expressed in the outer limiting membrane. 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.


ITGB8

CRB2

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. A mouse homolog has been found in the INL of mice. Our analysis shows that CABP5 is expressed in the INL and ganglion cells in the human retina. The axons of ganglion cells are collected in the nerve fiber layer, as exemplified by PI16, and extend through the optic nerve to various visual centers in the central nervous system. PI16 is a peptidase inhibitor that has been reported to be involved in stress response in cardiac muscle and vasculature. Its function in the retina is not yet characterized.


VSX2

RCVRN


CABP5

PI16

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 79 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.

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

Figure 3. An interactive network plot of the retina enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of retina enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.


Retina shares most group enriched gene expression with the brain (n=37), 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


Skin is another tissue that shares expression of several (n=6) group enriched genes with the retina. Tyrosinkinase (TYR) converts tyrosine to melanin. It is expressed in melanocytes in skin and hair, and in pigment epithelial cells in the retina.


TYR - retina

TYR - hair


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 108 genes have been analyzed in retina.

Gene Gene description Staining pattern
ABCA4 ATP binding cassette subfamily A member 4 Staining in photoreceptor cells.
ABCG4 ATP binding cassette subfamily G member 4 Staining in photoreceptor, plexiform and nerve fiber layers.
AC010319.2 Staining mainly in outer limiting membrane.
AIPL1 Aryl hydrocarbon receptor interacting protein like 1 Positivity in photoreceptor cells.
ANKRD33 Ankyrin repeat domain 33 Staining in photoreceptors.
ANO2 Anoctamin 2 Positivity in outer plexiform layer.
ARMS2 Age-related maculopathy susceptibility 2 Positivity in outer and inner plexiform layers.
ARR3 Arrestin 3 Staining in cones.
ATP1B2 ATPase Na+/K+ transporting subunit beta 2 Staining in most layers, strongest in photoreceptor cells and inner nuclear layer.
BCO2 Beta-carotene oxygenase 2 Staining in photoreceptor cells and pigment epithelium.
BEST1 Bestrophin 1 Positivity in basal membrane of retinal pigment epithelium.
CABP5 Calcium binding protein 5 Positivity in ganglion cells and cells in inner nuclear layer.
CACNA1F Calcium voltage-gated channel subunit alpha1 F Positivity in photoreceptor cells segments.
CDHR1 Cadherin related family member 1 Positivity in photoreceptor cell outer segments.
CFAP161 Cilia and flagella associated protein 161 Staining in cones and nerve fibers.
CHRNA3 Cholinergic receptor nicotinic alpha 3 subunit Positivity in cells in photoreceptor layer, nerve fibers in outer and inner plexiform layer and nerve fiber layer.
CLIC6 Chloride intracellular channel 6 Positivity in nerve fibers in nerve fiber layer.
CLUL1 Clusterin like 1 Positivity in rods.
CNGB3 Cyclic nucleotide gated channel beta 3 Positivity in outer liming membrane.
COL9A1 Collagen type IX alpha 1 chain Positivity in cones.
CPLX4 Complexin 4 Positivity in outer and inner plexiform layers.
CRABP1 Cellular retinoic acid binding protein 1 Positivity in inner nuclear layer and nerve fibers.
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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 that 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 provide nutrients and protection against UV-radiation 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.


Relevant links and publications

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

Yu NY et al., Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res. (2015)
PubMed: 26117540 DOI: 10.1093/nar/gkv608

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