Em busca de novos métodos de tratamento para a retinose pigmentar causada por mutações na rodopsina por Fernanda Balen - Versão HTML

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Vision is an important sensory model system for vertebrates since it most directly

mediates the interaction with the exterior world. Not surprisingly, the eye has evolved to be an

organ of extreme perfection and complexity. This was elegantly pointed out by Charles

Darwin, who said:

To suppose that the eye, with all its inimitable contrivances for adjusting the focus to

different distances, for admitting different amounts of light, and for the correction of

spherical and chromatic aberration, could have been formed by natural selection

seems, I freely confess, absurd in the highest possible degree. Yet reason tells me, that

if numerous gradations from a perfect and complex eye to one very imperfect and

simple, each grade being useful to its possessor, can be shown to exist; if further, the

eye does vary ever so slightly, and the variations be inherited, which is certainly the

case; and if any variation or modification in the organ be ever useful to an animal

under changing conditions of life, then the difficulty of believing that a perfect and

complex eye could be formed by natural selection, though insuperable by our

imagination, can hardly be considered real. How a nerve comes to be sensitive to

light, hardly concerns more than how life itself first originated; but I may remark that

several facts make me suspect that any sensitive nerve may be rendered sensitive to

light, and likewise to those coarser vibrations of the air which produce sounds

(Darwin, 2006). Charles Darwin 1809-1882.

The “perfection” of the eye can be well illustrated by the capability of adaptation that

the eye presents in front of specific needs of the organisms. Vision in humans has evolved to

accommodate both, daylight and night vision. Nocturnal animals have their visual systems

optimized for night activity. Deep sea animal vision is adapted to the limited radiation that

penetrates their habitat.

1.1.1 The vertebrate eye

The retina is a very complex and layered structure that lines the back of the eye

(Fig.1.1 and 1.2). It has a highly ordered anatomical organization, with a few basic classes of

cells located in the outer nuclear layer (rods and cones), inner nuclear layer (bipolar,

horizontal, and amacrine cells) and ganglion cell layer (ganglion cells).




Figure 1.1 - Structure of the eye.

SOURCE: Eye diagram is a courtesy of the National Eye Institute, National Institutes of Health. With


Figure 1.2 - Axial organization of the retina.

SOURCE: Drawing is adapted from (Cajal, 1900). With permission.

Figure 1.3 shows the three layers of neuronal cell bodies (outer nuclear, inner nuclear

and ganglion cell layers) interconnected by synapses in the two plexiform layers (outer and




Light must pass through the entire retinal thickness to reach the outer segments of

photoreceptors (rods and cones), where the phototransduction occurs. To avoid image

distortion and loss, Müller glial cells, whose cell bodies are located in the inner nuclear layer,

appear to act as living fiber optics (Franze et al., 2007).

Photoreceptor axons contact the dendrites of bipolar cells and horizontal cells in the

outer plexiform layer (OPL). In turn, the bipolar cells transmit the signal to ganglion cell

dendrites and amacrine cells in the inner plexiform layer. Last, the ganglion cells send their

axons through the optic fiber layer to the optic disk to make up the optic nerve (Molavi,

1997). Photoreceptors, bipolar, and ganglion cells release glutamate to mediate the

information from retina to the brain.

Figure 1.3 - Light micrograph of a vertical section through the human retina and a scheme identifying

the cells type.

SOURCE: Figure adapted from (Kolb, 2011). With permission.

The photoreceptor cells, rods and cones, are responsible for different parts of visual

perception. Rods, the most abundant photoreceptor cells, are responsible for black and white

vision under dim light conditions. Rod mediated vision is very sensitive to light, but do not

enable color vision. Cone mediated vision are responsible for day light vision. They present

high resolution and are sensitive to color and details.

Besides rods and cones, photosensitive ganglion cells containing melanopsin were also

described in mammals (Foster et al., 1991; Lucas et al., 2001; Provencio et al., 2000). They



are involved in various reflexive responses of the brain and body to the presence of light, for

example the regulation of circadian rhythms, the pupillary reflex and other non-visual responses to light.

Anatomically, vertebrate rods and cones photoreceptor cells contain an outer segment,

which carries the photosensitive pigments; an inner segment, a nucleus and a synaptic

terminal (Figure 1.4). The rod outer segment (ROS) is packed by a large number of discs,

where the visual photoreceptor molecule rhodopsin is located. Rhodopsin consists of the

apoprotein opsin and the, Vitamin A derivative, chromophore 11-cis retinal (Wald, 1968).

Figure 1.4 - Schematic structure of the rod photoreceptor.

SOURCE: Balen, 2012

1.1.2 Rhodopsin

The photoreceptor molecule rhodopsin is a membrane protein, and a prototypical

member of the large G protein-coupled receptor (GPCR) family, the largest family of cell

surface receptors. Rhodopsin (Figure 1.5) is made up of a cytoplasmatic domain (CP), a


seven-transmembrane helical domain (TM) and an extracellular domain (EC). Rhodopsin

contains a covalently linked ligand, 11-cis retinal, a Vitamin A derivative, that stabilizes the

folded receptor (Figure 1.5). Visual signal transduction is initiated when a photon induces

isomerization of 11-cis retinal to all-trans retinal. This event triggers the rearrangement of the

TM domain, resulting in the light-activated, Metarhodopsin II (Meta II) state. The Meta II

state of rhodopsin is the active functionally state of the protein and initiates the visual signal

transduction cascade. As a first step, Meta II binds to the G protein, transducin (Gt). Gt then

activates phosphodiesterase (PDE), which hydrolyzes cyclic GMP (cGMP). This event

ultimately leads to the hyperpolarization of the cell through the closure of ion channels. When

the cell is hyperpolarized, the electrical potential inside the cell is more negative than in

darkness. In contrast, in the dark state, cGMP keeps the channels in the photoreceptor

membrane open, and Na+-Ca2+ influx depolarizes the membrane. The signal can be shut down

through two mechanisms: (1) through Meta II decay, a process of dissociation of protein and

ligand to opsin and free all-trans retinal, or (2) through phosphorylation of Meta II by

rhodopsin kinase, followed by binding of arrestin to the phosphorylated C-terminal end of

rhodopsin (Chabre et al., 1988). The ligand free opsin formed when Meta II decays can

readily uptake 11-cis-retinal, thus regenerating rhodopsin.



Figure 1.5 - Tertiary structure of rhodopsin.

SOURCE: Balen, 2012

The primary structure of rhodopsin (bovine) contains 348 amino acids. This sequence

was determined using amino acid sequencing (Hargrave et al., 1983; Ovchinnikov Yu et al.,

1982). Figure 1.6 shows the secondary structure model of rhodopsin showing the seven TM

helices, the EC domain and the C-terminus domain, which faces the CP, along with the

disulfide bond (Cys110 and Cys187) and palmitoylation sites (Cys322 and Cys323). The

structure is divided in about 50% TM, 25% CP and 25% EC domains. Rhodopsin was the first

protein of the GPCR family to be crystallized (Palczewski et al., 2000). Rhodopsin it the best

studied and the prototypic member of the GPCR family.



Figure 1.6 - Secondary structure of rhodopsin.

SOURCE: Balen, 2012

Since the correct folding of the rhodopsin protein is extremely important for its

functionality, many studies were conducted to understand the folding and unfolding

mechanisms of rhodopsin. It has been demonstrated that there is a minimum amount of helix

content necessary to maintain the binding of 11-cis retinal to the protein (Ridge et al., 1995).

Unfolding studies using single molecule force spectroscopic approaches, point at specific

areas of rigidity in the structure of rhodopsin (Sapra et al., 2006, 2008; Tastan et al., 2007).

Furthermore , in silico simulations of thermal unfolding suggested formation of a folding core

between the EC and TM domains (Rader et al., 2004; Tastan et al., 2007). The structure of

rhodopsin is also prone to misfolding when amino acid replacements are made (Anukanth and

Khorana, 1994; Garriga et al., 1996; Kaushal and Khorana, 1994; Liu et al., 1996). Misfolding

here is characterized by the inability of the protein to bind 11-cis retinal, necessary for the

correct function of the protein in the visual system.


1.1.3 Allosteric ligands

In addition to retinal isomers, rhodopsin has been shown to bind a number of other

ligands. They all bind in locations other than the retinal binding pocket and are thus by

definition allosteric ligands. These ligands are reviewed below.


Zinc, after iron, is the second most abundant trace metal in the human body (Aggett

and Comerford, 1995). It is present in high concentrations in the eye, especially in the retina

(Grahn et al., 2001). Numerous normal retinal functions are dependent on Zn2+, including

retinal development, synaptic transmission, light response and Vitamin A metabolism (Grahn

et al., 2001). Zn2+ deficiency leads to night blindness, changes in dark-adaptation, ultra-

structural changes in the retina and the retinal pigment epithelium, and age-related macular

degeneration (Ugarte and Osborne, 2001). Furthermore, patients with the retinal degeneration

disease retinitis pigmentosa (see below) exhibit decreased serum Zn2+ levels (Karcioglu et al.,

1984). However, especially for age-related macular degeneration, opposing roles have been

proposed for Zn2+. While supplementation with Zn2+ is the most widely used intervention

strategy for patients with this disease, its presence in Drusen, deposits of accumulated

extracellular material containing misfolded proteins which especially occur in patients with

age-related macular degeneration, has been linked to increased aggregation of proteins

(Lengyel and Peto, 2008). There are probably multiple proteins responsible for these

physiological effects (Grahn et al., 2001), but one particularly well established target is the

photoreceptor rhodopsin, to which Zn2+ binds directly and specifically with milli- to

micromolar affinities depending on the membrane environment studied (Shuster et al., 1992).

The crystal structure of rhodopsin revealed four putative Zn2+ binding sites (Okada et al.,

2002; Palczewski et al., 2000), shown in Figure 1.7 as purple spheres. The extracellular and

transmembrane Zn2+ binding sites have been proposed to be the physiologically relevant ones

(Park et al., 2007; Stojanovic et al., 2004), but X-ray absorption spectroscopy (Toledo et al.,

2008), NMR spectroscopy (Patel et al., 2005) and proteolytic digestion (Shuster et al., 1992)

have suggested the primary binding sites to be located in the EC domain.



Figure 1.7 - Cartoon representation of dark state rhodopsin highlighting the presence of Zn2+ bound.

The four Zn2+ ions bound are represented as spheres and colored in magenta. Images were generated using VMD

visualization software (Humphrey et al., 1996).

SOURCE: The structure shown is PDB identification 1L9H (Okada et al., 2002; Palczewski et al., 2000).


Anthocyanins are plant pigments belonging to the large group of phenolic compounds,

the flavonoids. They are abundant in a diet rich in fruits and vegetables. Anthocyanin

consumption confers a broad spectrum of health benefits, especially in vision, where they

have been implicated to have protecting, enhancing and restoring abilities. They also have

anti-oxidant, anti-cancer, anti-diabetic, anti-inflammatory, anti-aging and cardioprotective

properties (Bagchi et al., 2004; Kaushal and Khorana, 1994; Lila, 2004; Zafra-Stone et al.,

2007). Recently, anthocyanins from blackcurrants were shown to enhance the regeneration of

rhodopsin, directly linking anthocyanin action to vision-related functional effects

(Matsumoto et al., 2003). In the dark, 11-cis retinal bound via Schiff base linkage to Lys296

in the TM domain of rhodopsin, gives rise to a chromophore with absorbance maximum at

500nm (Figure 1.8A). Illumination results in formation of the activated Meta II state of the

protein with characteristic absorbance maximum at 380nm (Figure 1.8A), in which the all-

trans retinal isomer is bound. Slowly – in the order of minutes – the Schiff base is

hydrolyzed and all-trans retinal leaves the protein, forming opsin. This Meta II decay can be



followed by tryptophan fluorescence (Figure 1.8B). The essay consists of illumination of the

sample, and measuring the rate of fluorescence increase, as the tryptophans are exposed,

corresponding to the rate of retinal release.

Opsin is then able to bind free 11-cis retinal and the 500nm chromophore is restored.

This process is referred to as regeneration. The faster rates of rhodopsin regeneration in the

presence of anthocyanins are potentially of functional importance because under dim-light

conditions it is critical to ensure that rhodopsin is quickly available after activation to

participate in a renewed visual signal transduction cascade.

Figure 1.8 - Application of different spectroscopic methods to the analysis of rhodopsin.

(A) Absorbance spectrum. For absorbance spectrum, rhodopsin in the dark (R; black line) and after illumination

(R*; grey line) are shown. The rhodopsin concentration is 0.5µM. (B) Fluorescence emission spectrum

(excitation wavelength 295nm) in the dark (R; black line) and 60min after illumination (R*; grey line). The

rhodopsin concentration is 0.5µM.

SOURCE: Figure adapted from (Tirupula et al., 2009). With permission.

Anthocyanin cyanidin-3-O-glucoside (C3G) binds directly to rhodopsin in the dark

and upon light activation, probably in its cytoplasmic domain (Yanamala, 2009). The

apparent affinity of C3G for dark-adapted rhodopsin is 50-100µM based on 1H NMR data

recorded at pH 6, but the affinities for light-activated and opsin states is likely higher and

remain to be determined. Evidence was provided that the mode of interaction not only varies

with activation-state but also with pH, as C3G exists in multiple chemical forms, namely the

flavylium ion, the quinoidal base, the carbinol pseudobase and the chalcone, which are

shown in Figure 1.9 (Veitch and Grayer, 2008).




Figure 1.9 - Chemical structures of pH dependent equilibrium species of the cyanidin-3-glucoside.

(A) Flavylium cation. (B) Quinoidal base example. (C) Carbinol pseudobase. (D) Chalcone. „R‟ in the chemical

structures of C3G represents the glucoside sidechain.

SOURCE: Figure adapted from (Yanamala et al., 2009). With permission.

Chlorin e6

Porphyrins are an omnipotent class of naturally occurring compounds with many

important biological representatives such as hemes and chlorophylls. The chlorophyll

derivative chlorine e6 (Ce6) (Figure 1.10) is believed to enhance sensitivity of rhodopsin to

red light in deep-sea fish rhodopsin (Douglas et al., 1999; Isayama et al., 2006).

Figure 1.10

- Chemical structure of Ce6.

SOURCE: Balen, 2012

In vivo studies with salamander and mouse models have confirmed that Ce6 can

effectively enhance vision in these animals (Washington et al., 2007). A decrease in the

500nm peak of a UV-Vis spectrum of salamander rhodopsin was observed by illuminating the

sample at 668nm, a wavelength at which Ce6 absorbs while rhodopsin has less response

(Isayama et al., 2006). Additionally, mice administered with Ce6, showed a two-fold increase

in electroretinogram b-wave amplitudes (the mass response to the eye) as a response to red

and blue light (Washington et al., 2007). An energy transfer between Ce6 and 11-cis retinal


has been proposed (Isayama et al., 2006). In vitro studies demonstrated that Ce6 physically

binds to rhodopsin and modulates its structure and function (Yanamala, 2009).

1.1.4 Retinitis pigmentosa

Mutations in rhodopsin are associated with the retinal degeneration disease retinitis

pigmentosa (RP). RP reflects a large group of genetically heterogeneous disorders that affect

the photoreceptors and retinal pigment epithelium (RPE) diffusely across the entire fundus but

begin with initial geographic involvement in either the periphery or the macula (Sieving,

2010). It is one of the major causes of blindness in the world, affecting 1 in 4000-5000 people

(Kannabiran, 2008). The disease is marked by early rod photoreceptor dysfunction and

progressive degeneration of rods, which impairs vision in dim light and causes loss of

peripheral vision, that is “tunnel vision” (Sieving, 2010), and later cones (Lam, 2005). This

sequence of events explains why patients initially present night blindness, and only later in

life presents diurnal visual impairment (Hamel, 2006). Typical RP is then described as rod-

cone dystrophy, where rods are more affected than cones. However, some of the allied forms

primarily cause cone photoreceptor loss and initially manifest with a reduction in central

visual acuity (Sieving, 2010).

The pathologic mechanism underlying the observation that at the latest stages in the

disease retinal degeneration is observed is still not clear. Family-related retinal degeneration

with intraneural retinal pigmentation was described as early as 1855 (Sieving, 2010), and

although “retinitis” implies an inflammatory or infectious cause, histopathology studies shows

no evidence of macrophage invasion or other inflammatory response in the photoreceptor

layer or elsewhere in the retina (Sieving, 2010). Studies attributed a genetic basis to the

majority cases of RP and recently concluded that apoptosis is the last stage contributing to

photoreceptor cell death (Chang et al., 1993; Portera-Cailliau et al., 1994; Reme et al., 1998).

Belmonte et al. (2006) showed that Rac1, a member of the GTPase family with very low

molecular weight, may participate in the cell death process when the degeneration is mediated

by light or inherited. Moreover, after degeneration, there was an increase in the activity of

Rac1, and furthermore Rac1 expressing photoreceptors were shown to be TUNEL-positive.

TUNEL is a common method for detecting DNA fragmentation, by labeling the terminal end of nucleic acids, which results from apoptotic signaling cascades.


No evidence of racial or ethnic predisposition has been observed. However, men may

be affected slightly more than women because of X-linked conditions (Sieving, 2010). The

age-at-onset of symptoms and diagnosis is variable and can range from early childhood to

mid-age adults. The diagnostic criteria is assessed through 4 parameters (Hamel, 2006); 1)

Functional signs: which are characterized by night blindness as the first symptom, followed

by photophobia; visual acuity is preserved in early and mid ages. 2) Visual field: marked by

patchy losses of peripheral vision evolving to ring-shaped scotoma, tunnel vision is the last

step. 3) Fundus: presence of pigmentary deposits similar to bone spicules in the peripheral

retina; thinning of the retinal vessels; waxy pallor of the optic nerve; various degrees of retinal

atrophy. 4) Electroretinogram: decrease of the amplitudes of a- and b- waves; scotopic

system, rods, predominates over photopic system, cones.

Methods for clinical diagnosis are based on the presence of night blindness and

peripheral visual defects, lesions in fundus, ERG changes, and progressive deterioration of all

of these signs (Hamel, 2006). The ERG is the best method to detect the disease, since patients

can be asymptomatic, for example presenting only nigh blindness and no other clinical signs.

1.1.5 Types of retinitis pigmentosa

RP is extremely heterogeneous, both clinically and genetically. Cases where there are

no family histories evident are known as isolate, sporadic or simplex. By inheritance, RP can

be classified in autosomal dominant (AD) (30%), autosomal recessive (AR) (20%) and X-

linked recessive (XL) (15%) or sporadic/simplex traits (30%), and be part of the group of

Leber congenital amaurosis (5%). These are among the more common modes of inheritance

in RP and the relative prevalence of each form varies between populations (Kannabiran, 2008;

Musarella and Macdonald, 2011). There are also rare forms of RP that are called X-linked

dominant, mitochondrial and digenic (presents mutations in two different genes) (Musarella

and Macdonald, 2011). Clinically, they are known as non-syndromic RP. Mutations in 16

different genes were found to cause autosomal dominant retinitis pigmentosa (ADRP)

(Kannabiran, 2008). Autosomal dominant forms are usually the mildest forms, some cases

starting after the age of 50, although severe disease can also appear (Hamel, 2006). The most

frequent cause of ADRP is a mutation found in the rhodopsin gene, affecting 20-25% of the

patients, especially in North America and Europe. In North America, a single point mutation,

Pro23His, accounts for 12% of the cases, although not significantly in Europe. Mutations on


the RDS gene is the second most predominant set of mutations in RP, 3.5 to 9% were

estimated followed by the RP1, which was estimated to be 4-8% of the cases. Other mutations

appear to be less frequent and are implicated in less than 5% of the cases (Kannabiran, 2008).

Autosomal recessive retinitis pigmentosa (ARRP) is a group of disorders that includes

juvenile and early-onset forms of RP. This group overlaps with another neurodegenerative

disease called Leber‟s congenital amaurosis (LCA). 21 genes were identified to cause ARRP.

However, the frequency of mutations from all of these genes accounts for approximately a

third of ARRP patients, with individual genes contributing to less than 5% of disease

(Kannabiran, 2008). There are two genes that were found to cause X-linked RP, RP2 and RP3

(known as RP GTPase regulator ( RPGR)). RP2 account for 10-20% of recessive XLRP and

mutations in RPGR account for 50-80% of recessive XLRP. Another six loci were also

mapped but the genes were not identified yet (Kannabiran, 2008). X-linked forms also start

early and are frequently associated with myopia. Transmission of the disease is recessive in

most cases; however there are some families in which dominant inheritance with affected

females is found (Hamel, 2006).

Many syndromes are associated with different types of pigmentary retinopathies, and

are known as syndromic RP. The most frequent are the Usher and Bardet Biedl syndromes

(BBS). Usher syndrome is the most frequent and is typically associated with neurosensory

deafness. BBS is marked by obesity (already in childhood), mental retardation or mild

psychomotor delay, post axial polydactyly, hypogenitalism and renal problems and RP

associated, frequently as cone-rod type of dystrophy (Hamel, 2006). Other less frequent

syndromes encompass those that cause renal abnormalities such as Senior Loken syndrome

(SLS) and Alport syndrome, dysmorphic syndromes such as Cohen, Jeune and Cockayne

syndromes, metabolic syndromes such as Methylmalonic aciduria with homocystinuria,

Abetalipoproteinemia (Bassen Korntzweig disease), Bietti‟s disease, Cystinosis,

Mucopolysaccharidoses, Zellweger (cerebro-hepato-renal) syndrome, Hyperoxaluria type I

with retinal atrophy in spots, Neonatal adrenoleukodystrophy with leopard spots fundus,

Infantile Refsum disease, Adult Refsum disease, Peroxisomal disorders other than Refsum

disease, Peroxisomal disorders other than Refsum disease and neurological disease, neuronal

ceroid lipofuscinosis, Joubert syndrome, autosomal dominant cerebellar ataxia type II

(SCA7), Myotonic dystrophy and Hallervorden-Spatz syndrome (Hamel, 2006).


1.1.6 Current therapies

Although innumerous studies have been performed, there is no effective therapy to

treat RP. Meanwhile, many studies are focusing on alternative therapies to prevent, treat or

slow down the disease.

In order to slow down the degeneration process, light protection and treatment with

vitamins is recommended. Animal studies suggested that light exposure was damaging for the

transgenic photoreceptors (Wang et al., 1997), and it is recommended to patients with

pigmentary retinophaties to avoid direct light exposure, by either wearing dark glasses or

yellow-orange spectacles, which minimizes photophobia. The use of eye shades and lateral

protection is also recommended to protect against light coming from the sides (Hamel, 2006).

Treatment with vitamins A and E are believed to protect the photoreceptors by trophic and

antioxidant effects. A clinical trial of nutritional supplements for adults diagnosed with the

common forms of RP was conducted (Kupfer, 1993). The course of retinal degeneration was

monitored by electroretinogram recordings. On average, patients taking 15,000 IU of vitamin

A daily showed slower, and those taking 400 IU of vitamin E showed faster retinal

degeneration. The molecular mechanism of the Vitamin A effect is the rescue of correctly

folded rhodopsin by retinal. However, 15,000 IU only slowed and could not prevent retinal

degeneration, and a daily vitamin A intake exceeding 25,000 IU over the long-term is toxic.

Later, similar studies were conducted and showed complementary or adverse results (Berson

et al., 1993a, 1993b, 2004, 2010).

Different therapies have therefore been explored (Kannabiran, 2008). Gene therapy

methods have been geared towards correction of the specific gene mutated, by means of gene

replacement for recessive gene mutations or suppression and replacement of mutant mRNAs

for dominant gene mutations. Encapsulated cells releasing neurotrophic factors have been

implemented with the aim of promoting survival and growth of photoreceptors. Anti-

apoptotic factors have been introduced by gene-delivery systems. Finally, stem cell

transplantation has been considered. Each approach is briefly out-lined below.

Gene therapy is a promising therapy for many inherited human diseases. The

technique requires efficient genotyping methods for identification of the implicated gene

(Hamel, 2006). Ocular gene therapy (OGT) has been successfully tried in different animals

and humans. First, identification of the genetic cause of the RP and genotyping of the patients

was performed in order to proceed with the genetic modification of mutant ocular cells to


produce therapeutic effects. The strategy of the therapy differs accordingly with the type of

disease or mutation target, and was already applied successfully in mouse (Bennicelli et al.,

2008; Tan et al., 2009), rats (Drenser et al., 1998; Lewin et al., 1998; Weber et al., 2003),

dogs (Acland et al., 2001; Veske et al., 1999; Weber et al., 2003), non-human primates

(Weber et al., 2003), and humans (Bainbridge et al., 2008; Hauswirth et al., 2008; Maguire et

al., 2008, 2009). Due to different outcomes from patients, more tests still need to be done in

order to prove the efficacy for all patients.

Retinal implants using either epiretinal or subretinal implants is also a good therapy

and have shown benefits in many tests with RP patients (Caspi et al., 2009; Humayun and de

Juan, 1998; Yanai et al., 2007) and animals models (Chow and Chow, 1997; Zrenner et al.,

1997). Clinical trials in human patients are already in Phase II, but still being improved.

Neurotrophic factors showed protective effects on photoreceptors degeneration,

including neurotrophic factor (CNTF), Glial cell-line derived neurotrophic factor (GDNF),

cardiotrophin-1, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor

(bFGF) (Hamel, 2006; Musarella and Macdonald, 2011) in animals (Bok et al., 2002; Frasson

et al., 1999; Li et al., 2010; Liang et al., 2001; Tao et al., 2002) and are in the trial phase in

humans (Sieving et al., 2006). Phase I and II are currently underway for treatment of atrophic

macular degeneration and RP.

Retinal transplantation consists of the transplantantion of sheets of retinal cells, layers

of photoreceptors or a complete retina (Hamel, 2006; Musarella and Macdonald, 2011).

Alternatively, transplantation of photoreceptor precursors has also been tried (Musarella and

Macdonald, 2011). Transplants in animal models and humans showed that transplanted

material does survive but not all presented evidence of a therapeutic effect (Gal et al., 2000;

MacLaren et al., 2006; Radtke et al., 2008).

Stem cell treatment is being used to regenerate degenerated retinas, based on its

capability to give rise to specialized cells and also its capability of self-renewal (Lund et al.,

2006). The donor cells and the host recipient are two major aspects of the procedure (Rivas

and Vecino, 2009). Hippocampal, embryonic and bone marrow cells have been tested in

models of retinal degenerative disorders and have showed high migratory and differentiation

capacity. In contrast, the use of retinal stem/progenitor cells in suspension or as neurospheres

exhibit poor migration, but were successful in expressing retina specific markers after

transplantation. Although progress is being made, extrapolation to humans is yet to be



Neuroprotection using antiapoptotic factors or inhibitors of apoptosis is also being

explored (Hamel, 2006; Kannabiran, 2008). Studies in mouse and rat models have shown

protection of photoreceptor cells (Bennett et al., 1998; Leonard et al., 2007; Liu et al., 1999;

Petrin et al., 2003).

1.1.7 Rhodopsin as a target in retinitis pigmentosa

The causes for RP are heterogeneous, and mutations in many different genes can lead

to RP. The first of these genes that has been identified as a genetic cause for RP was the rod

photoreceptor rhodopsin. Now more than 150 mutations in rhodopsin have been identified in

different RP patients (Cooper et al., 2007). From these, 92 are single point mutations in 64

amino acid positions (Figure 1.11). This is a remarkable number considering that rhodopsin is

a protein with a total length of only 348 amino acids. 42 of these RP mutations have been

studied in vitro and 31 of them (>70%) were found to cause misfolding of rhodopsin.

Many studies have focused on understanding the molecular mechanism underlying

rhodopsin mutations leading to RP development. The majority of the mutations studied

previously focused on the EC and TM domains of rhodopsin (Dryja et al., 1993, 1995). Most

of these mutations mediate formation of an incorrect disulfide bond, leading to an incorrectly

folded protein that cannot bind 11-cis retinal (Kaushal and Khorana, 1994; Ridge et al., 1995).

This finding suggested a structural coupling between the EC and TM regions, which is

important for the correct folding of rhodopsin.

Since the discovery of misfolding of rhodopsin as one of the leading causes for RP

(Kaushal and Khorana, 1994), much effort has concentrated on rescuing correctly folded

rhodopsin by adding excess Vitamin A or retinoid derivatives. Recently, a clinical trial of

nutritional supplements for adults with common forms of RP was conducted, and

demonstrated that vitamin A treatment alone cannot cure RP (see above).



Figure 1.11 - Secondary structure model of rhodopsin showing the positions of the mutations that cause

RP and congenital stationary night blindness.

SOURCE: Balen, 2012

1.1.8 Animal models of retinitis pigmentosa

Hereditary retinal degeneration affects human and other animals. The range of

diseases that affect animals can be due to natural occurrence or obtained through genetic

manipulation. These various mutations are of great importance as model system for the study

of the disease evolution, pathogenesis and strategies for treatment. Specifically for RP, there

are many animal models available and many of them mimic the disease that is found in


The list of animal models that present mutations that occur naturally includes dogs,

cats and chicken. Natural and induced mutations are studied in mouse, rats and pigs. These

animal models were reviewed recently (Rivas and Vecino, 2009) and are summarized here.

Canine animal models of RP are considered the best models due to the similarity

between dog and human eye disorders. Furthermore, since eye anatomy and size in dogs is

similar to human, the same apparatus for physical studies can be used for dogs as that for

humans. All canine diseases present rod-cone degenerations, where rod disease, dysfunction,

and death precede the loss of cones. These diseases are analogous to human RP and are called

Progressive Retinal Atrophy (PRA). The genes mutated include PDE6A and PDE6B encoding


the α and β subunits of the cGMP specific phosphodiesterase (autosomal recessive RP),

RPE65 gene (leber congenital amaurosis), the RP GTPase regulator RPGR (X-linked RP) and

rhodopsin RHO (autosomal dominat RP). A list of available canine models is provided below:

 erd - canine early retinal degeneration

 rcd-1 - rod-cone dysplasia type 1 - and rcd-2 - rod-cone dysplasia type 2

 RHO - rhodopsin, visual pigment of rod photoreceptor

 RPE65 – membrane-associated protein

 RPGR - mutations in the RP GTPase regulator

 RPGRIP1 – mutations in the RP GTPase regulator-interacting protein 1

Cats are good models too since they have a manageable eye size for examination and

manipulation. Their visual neurophysiology is well characterized and their eyes are

physiologically and anatomically similar to human. There are only two forms of feline PRA

that are well studied:

 rdAc - autosomal recessive rod-cone degenerative disorder

 Rdy model - autosomal dominant early-onset retinal degenerative disease

Chicken eyes are also useful to study since they can be compared with human eyes in

size, facilitating pathological examination. However, chicken eyes are very different from

human eyes from a number of perspectives. In particular, the chicken eye is cone-dominated

rather than rod-dominated. However, the conservation of gene order between chicken and

humans is similar to that between human and mice genome, making this a potential model for

the study of spontaneously occurring inherited blindness disease. There are two models of

chicken that presents retinal degeneration:

 rd – retinal degeneration chicken strain

 rdd – retinal dysplasia and degeneration

Mice are the most studied animal models, and the rod-less mouse was the first animal

model where retinal degeneration was described. Mouse and human have a genomic similarity

of about 90%, and gene correlation can be precisely achieved. Advantages in the use of mouse


include the short life span (about 2 years), availability of protocols that are well established

and the ease at which new single gene diseases can be mapped. Available mouse models are:

 Peripherin rds – retinal degeneration slow, rds-peripherin null mutation

 Rd – retinal degeneration, which includes rd1, rd4, rd8, rd10, rd12

 CEP290: rd16 – novel centrosomal protein

 I-255/256 – mutant opsin gene with 3-bp deletion of isoleucine at codon 255/256

 Knockout RPE65 (RPE65-/-) - accumulates retinyl esters in the RPE and lacks 11-cis


 P347S - proline-347 to serine mutation in rhodopsin

 Peripherin-rds 307 – mice heterozygous for the codon 307 mutation

 Q334ter - transgenic mice that express a truncated rhodopsin due to a mutation

resulting in a stop codon at position 344

 Knockout rho (rho-/-) – mutations in the rhodopsin gene (have no functional

rhodopsin gene)

 VPP – mice express a mouse opsin gene containing three point mutations within a

seven amino acid sequence near the N-terminus of the molecule

o P23H - histidine for proline mutation

o V20G – glycine for valine mutation

o P27L – leucine for proline mutation

Rats exhibit retinal degeneration very similar to the retinal degeneration observed in

humans. The benefits of using rat photoreceptors are that they have been well studied,

therefore extensive knowledge of basic biological mechanisms is known. Morphologically,

the rat eye is several times larger than the mouse, which simplifies surgical manipulations and

electroretinographic evaluation; they also breed as rapidly as mice, generating large litters in a

short gestational time. The following lines are available:

 RCS – Royal College of Surgeons. Retinal pigment epithelium (RPE) is unable to

phagocytose shed photoreceptor outer segments.

 P23H – histidine at proline 23 position in rhodopsin. Rat carries a mutant mouse opsin

gene in addition to the endogenous native opsin genes


 S334ter – the opsin transgene contains a termination codon at residue 334, resulting in

the expression of a rhodopsin protein lacking the last 15 C-terminal amino acids

Pigs are genetically engineered animal models. Pig eyes are similar in size to the

human eyes. They have a similar number and distribution of rod and cone cells as humans,

which makes them an excellent model for RP. Among other large mammals, the porcine

retina is even more similar to the human retina in size, and tools applied for diagnostics can

be used without adaptations.

 P347L - lysine for proline at position 347 in rhodopsin

 P347S - serine for proline at position 347 in rhodopsin

The figure below represents the time of initiation of retinal degeneration in each

animal model (Figure 1.12). Knowledge of the different degeneration times is important in

order to decide which model is more relevant to specific studies such as conducted as part of

this thesis.



Figure 1.12 - Schematic representation of the initiation of the retinal degeneration in each animal model.

Continuous lines: normal retina (non-degenerating); discontinuous lines: degeneration of the retina; Red:

morphological states; Blue: electroretinographic state; each square represents a postnatal age of the animal


SOURCE: Figure is taken from (Rivas and Vecino, 2009). With permission.



1.2.1 Open questions

Previous studies by Khorana and coworkers have addressed the identification of the

amino acids stabilizing rhodopsin and have established that there is tight coupling between the

extracellular, transmembrane and cytoplasmic domains in rhodopsin (Khorana, 2000).

Disruption of this coupling causes irreversible misfolding. Recent simulations of misfolding

of the rhodopsin tertiary structure have provided a mechanistic understanding of these

findings (Rader et al., 2004). A major core of residues contributing to rhodopsin stability was

identified at the interface between the extracellular and transmembrane domains. A small

additional region at the conserved DERY motif in the cytoplasmic domain was also

implicated in playing a role in rhodopsin stability. More than 90% of the residues in the large

core cause misfolding upon mutations (Rader et al., 2004). Furthermore, recent findings with

RP patients and molecular studies have correlated these molecular studies with RP patients

studies (Iannaccone et al., 2006). This indicates that it may be possible to develop new

avenues for RP treatments based on stabilization of rhodopsin structure. Such new avenues

are needed because there is, so far, still no cure or treatment for RP.

Here we explore novel approaches for the stabilization of opsin/rhodopsin through

small molecules. Rhodopsin has been shown to be a receptor for anthocyanins (Lila, 2004)

and its spectral range can be extended by interacting with porphyrin compounds (Washington

et al., 2004) . Members of the flavonoid group of phytochemicals, anthocyanins are mostly

found in teas, honey, wines, fruits, vegetables, nuts, olive oils, cocoa and cereal.

Anthocyanins pigments and the flavonoids associated are known to protect against many

kinds of diseases. The enhancement of night-time vision by anthocyanins is known (Lila,

2004). Porphyrins are an omnipotent class of naturally occurring compounds with many

important biological representatives such as hemes and chlorophylls. Previous evidence from

our laboratory suggests that porphyrin and anthocyanin compounds both stabilize rhodopsin

and/or opsin (Yanamala, 2009).

In addition, studies have shown that providing patients with more retinal, via

administration of Vitamin A, can only partially slow down RP. So far, many alternative

approaches have been proposed, but none have an entirely positive effect on reversing the


disease. The hypothesis that molecules other than retinal can alone or in conjunction with

retinal rescue folded rhodopsin may lead to new treatments of RP.

1.2.2 Objectives and Approaches

This thesis aims to investigate the hypothesis that molecules other than retinal can

rescue folded rhodopsin and/or reduce photoreceptor cell death, which could potentially lead

to new treatments for RP. To address the open questions, the following specific aims are


Specific aim 1: In vitro molecular characterization of WT and different rhodopsin


A study comparing two RP mutations N15S and P23H with WT rhodopsin was

performed. Rhodopsin and mutants were expressed in COS-1 and HEK-293 cells followed by

purification after reconstitution with 9 -cis or 11 -cis retinal, during or after expression in the

cells. This provided a quantitative understanding of the role of retinal in the rescue of folded

rhodopsin. Using thermal denaturation, Total Reflectance Fourier Transform Infrared

(ATR/FT-IR) and fluorescence spectroscopy, SDS-PAGE mobility, glycosylation studies, and

subcellular localization using confocal microscopy, a quantitative comparison of molecular

properties of the mutants as compared to the WT was assessed.

Specific aim 2: Effects of ligand binding on rhodopsin structure and stability

First, stability studies using the protein rhodopsin from native source was carried out.

Next, the effect of different compounds such as metal ions, porphyrins and anthocyanins were

tested in order to assess their effect on the stability of rhodopsin. UV-visible, fluorescence and

CD spectroscopy were used in order to compare of molecular properties of the WT rhodopsin

alone and in presence of the different ligands tested.

Specific aim 3: In vivo drug treatment of light-damaged and mutant rats with

hereditary RP disease

The aim was to test whether Ce6 is able to prevent apoptosis in Sprague Dawley rats

submitted to light-induced degeneration, and in P23H and S334ter rat models of RP.

Morphological and functional (ERG) analyses were performed.