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Novel Aspects of Neuroprotection, 2010: ISBN: 978-81-308-0394-4
Editors: Francisco Javier Romero Gómez, Maria Miranda and Jose Miguel Soria
10. Antioxidant therapy in retinitis
pigmentosa
1
María Miranda1, Raquel Alvarez-Nölting1, Araiz J2
and Francisco Javier Romero Gómez1,3
Universidad CEU-Cardenal Herrera. Departamento de Fisiología, Farmacología
y Toxicología. Avda Seminario s/n, Moncada, Valencia, Spain; 2Department of
Ophthalmology, University of the Basque Country, Spain; 3Fundación
Oftalmológica del Mediterráneo (FOM), Bifurcación Pio Baroja-General Avilés
s/n 46015 Valencia, Spain
Abstract. Increasing evidence suggests that oxidative stress
contributes to the pathogenesis of many neurodegenerative
disorders, including retinitis pigmentosa (RP), age related macular
degeneration (AMD), glaucoma, diabetic retinopathy, and light
damage. RP refers to a group of diseases in which a mutation
results in death of rod photoreceptors followed by gradual death of
cones. In this review we focus on the importance of oxidative
stress alterations in animal models of RP and the possibility of
using antioxidants as a new strategy to delay photoreceptor
degeneration. It has been suggested that glutathione alterations can
also be observed in humans affected with photoreceptors
dystrophy. In addition, microarray experiments have shown that in
the rd1 retina, genes for products related to protection against
oxidative stress are up-regulated when compared to wild type mice.
Several studies have shown that the use of antioxidants, in vitro
Correspondence/Reprint request: Dr. Francisco Javier Romero Gómez, Universidad CEU-Cardenal Herrera
Departamento de Fisiología, Farmacología y Toxicología. Avda Seminario s/n, Moncada, Valencia, Spain
E-mail: [email protected]
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Maria Miranda et al.
and in vivo delayed the degeneration process significantly. Among the antioxidants
used in these studies are: zeaxanthin, lutein, α-lipoic acid and glutathione,
(α-tocopherol, ascorbic acid, Mn(III) tetrakis (4-benzoic acid) porphyrin,
docosahexaenoic acid and melatnin. Unless the common upstream initiator for a
photoreceptor dystrophy is found, multiple rescue paradigms need to be used to target
all active pathways. Furthermore, as the mammalian antioxidant defence system is a
complex network and comprise several enzymatic and non-enzymatic entities, it
appeared reasonable to combine different antioxidants instead of using them
individually.
1. Retinitis pigmentosa and its relation with oxidative stress
Retinitis pigmentosa (RP) refers to a group of diseases in which a
mutation results in death of rod photoreceptors followed by gradual death of
cones. Mutations in 36 different genes have been found to cause RP,
and mutations in many more cause widespread rod cell death in
association with syndromes that have extraocular manifestations
(www.sph.uth.tmc.edu_RetNet_sum-dis.htm). The enormous genetic
heterogeneity among the diseases that constitute RP is a problem for the
development of treatments that deal with primary genetic defects.
Despite the diversity of retinal degeneration disorders, apoptosis of
photoreceptors seems to be a feature common to all [1-3]. The signalling
pathways of apoptosis in photoreceptor cell death are still not fully
understood. Several studies have demonstrated that the eye is particularly
sensitive to oxidative damage. Because of its high oxygen request and
content of unsaturated lipid and its constant exposure to light retina may be
an elective site for oxygen radical production and lipid peroxidation [4-7].
After the death of rods, cone photoreceptors begin to die. The mechanism
of the gradual death of cones is one of the key unsolved mysteries of RP.
Another possible explanation for the slowly progressive death of cones after
the death of rods is oxidative damage. Rods are more numerous than cones
and are metabolically active cells with a high level of oxygen consumption.
As death of rods occurs, the level of oxygen in the retina increases [8, 9].
It has also been demonstrated an increased expression of genes involved
in cell proliferation pathways and oxidative stress at PN14 (the peak of rod
degeneration), at PN35 (early in cone degeneration) and at PN50 (during
cone degeneration) [10]. Carnody et al [11] demonstrated an early and
sustained increase in intracellular reactive oxygen species accompanied by a
rapid depletion of intracellular glutathione in an in vitro model of
photoreceptor apoptosis and that these early changes in the cellular redox
state precede disruption of mitochondrial transmembrane potential, nuclear
Antioxidant therapy in retinitis pigmentosa
3
condensation, DNA nicking, and cell shrinkage, all of which are wellcharacterized events of apoptotic cell death.
Moreover, the utility of an antioxidant, like CR-6, has been proved to
block photoreceptor apoptosis in vitro, probably by preventing the activation
of a pathway in which calpains have a key role [12].
The rd1/rd1 mouse has an insertion of viral DNA in the β-subunit of the
cGMP phosphodiesterase gene [13]. The mutation leads to toxic accumulation
of the second messenger cGMP and subsequent abnormally high Ca2+ levels
in the rd1 photoreceptors [1, 14]. This leads to an apoptotic-like rod cell
death [15, 16], followed by a mutation independent cone death. A mutation
in the same gene has been found in human forms of autosomal recessive
retinitis pigmentosa making the rd mouse retina an ideal model for
experimental analysis of human retinal dystrophies [17]. Moreover, oxidative
stress is a common product of ionic imbalance or elevated Ca2+ levels and it
has been found to precede calpain and caspase-3 activation in the rd1 mouse
retina [18]. All these findings provide targets for future treatment strategies.
2. Glutathione metabolism
Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most
abundant low-molecular-weight thiol, and GSH/glutathione disulfide is the
major redox couple in animal cells. It is formed in a two-step enzymatic
process including, first, the formation of gamma-glutamylcysteine from
glutamate and cysteine, by the activity of the gamma-glutamylcysteine
synthetase; and second, the formation of GSH by the activity of GSH
synthetase which uses gamma-glutamylcysteine and glycine as substrates.
While its synthesis and metabolism occurs intracellularly, its catabolism
occurs extracellularly by a series of enzymatic and plasma membrane
transport steps [19]. Glutathione is the most important and effective
endogenous antioxidant. It is considered as the body’s first line of defense
against oxidative stress.
It is a small molecule found in almost every cell. It cannot enter most
cells directly and therefore must be made inside the cell, from its three
constituent amino acids. Compelling evidence shows that GSH synthesis is
regulated primarily by gamma-glutamylcysteine synthetase activity, cysteine
availability, and GSH feedback inhibition.
Protein S-glutathionylation, the reversible binding of glutathione to
protein thiols (PSH), is involved in protein redox regulation, storage of
glutathione, and protection of PSH from irreversible oxidation [20].
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Maria Miranda et al.
Figure 1. Chemical structure of glutathione.
Among the roles attributed to GSH are maintenance of protein sulfhydryl
groups; protection of cells against oxidative or radiation-induced damages;
detoxification of highly reactive xenobiotic metabolites or peroxides; and
regeneration of antioxidant vitamins. GSH, ascorbic acid, and vitamin E
special is that they interact in a series of coupled oxidation-reduction
reactions. GSH is capable of reducing dehydroascorbic acid back to AA,
which, in turn, reduces oxidized vitamin E. GSH is normally regenerated
from the oxidized disulfide (GSSG) by a mechanism involving the NADPHdependent glutathione reductase. More recent studies of the functions served
by GSH in cells include modulation of protein function via thiolation which
may control physiological and pathophysiological pathways to include DNA
synthesis and repair, protein synthesis, amino acid transport, modulation of
glutamate receptors and neurohormonal signaling [21].
Light and oxygen are essential for vision, but paradoxically these
elements also trigger the formation of reactive oxygen species (ROS).
Because the eye is continuously exposed to ambient light energy, highly
efficient retinal defense mechanisms act to protect against photoinduced
damage. It is known that GSH is present in the retina and that this tissue also
has enzymatic activity associated with GSH metabolism like glutathione
peroxidase, glutathione disulfide reductase, and glutathione S-transferase.
Furthermore, continuous phagocytosis and degradation of retinal
photoreceptor outer segment material by the retinal pigment epithelium
(RPE) mitigates lipid peroxidative damage [22].
Despite this, it has been reported no GSH immunoreactivity in outer
segments of rod and cone photoreceptors from rodent, primate and zebrafish
retinas[23-25], but , Müller cells and inner retinal neurons appear to contain
substantial pools of this compound [26].
Each cellular compartment contains different pools of thiol/disulfide
couples, such as glutathione (GSH)/glutathione disulfide (GSSG), cysteine
(Cys)/cystine (CySS), and dihydrolipoic acid/lipoic acid [27]. The functions
of these redox couples are dependent on both the ratio and total
concentrations of the reduced and oxidized forms, the inherent electron
Antioxidant therapy in retinitis pigmentosa
5
donating/accepting characters, and the kinetics of interactions of the
components. Changes in the thiol/disulfide redox state alter signal
transduction, DNA and RNA synthesis, protein synthesis, enzyme activation,
and cell cycle regulation [28, 29].
2.1. Glutathione and nitric oxide
Nitric oxide (NO) has also been implicated in neurodegenerative diseases
[30]. NO is generated in the central nervous system by three isoforms of
nitric oxide synthase (NOS) located in the endothelial cells, astroglias, and a
few neurons [31]. NO plays an important role in cell-to-cell modulation and
vasodilatation via activation of NO-sensitive guanylyl cyclase and the
generation of cGMP [31]. Since the measurement of the NO radical by itself
is difficult because it is a radical with poor stability and with a very short
half-life, measurement of the end products of NO as nitrite and nitrate
(NO2−/NO3−) is often used as a marker for the production of NO radicals.
Modifications of the cellular redox state of the eye are believed to
contribute to the pathogenesis of many diseases and it has been demonstrated
that nitric oxide and reactive oxygen species (ROS) are key signalling
molecules in driving apoptosis both in in vitro and in vivo models of retinal
disease [12].
Recently, Komeima et al [32] demonstrated that peroxynitrite-induced
nitrosative damage also occurs in the rd1 mouse model of RP, because they
found an increase in S-nitrosocysteine and nitrotyrosine protein adducts that
are generated by peroxynitrite. They also found that treatment of rd1 mice
with a mixture of nitric oxide synthase (NOS) inhibitors markedly reduced
S-nitrosocysteine and nitrotyrosine staining and significantly increased cone
survival, indicating that NO-derived peroxynitrite contributes to cone cell
death.
NO may interact with oxygen, superoxide anion, and thiol compounds,
generating reactive nitrogen species (NOx), peroxynitrite, and S-nitrosothiols
including S-nitrosoglutathione (GSNO) [33]. These NO-derived species may
produce biological functions either similar or opposite to that of NO. For
example, peroxynitrite may cause oxidative stress and possibly neurotoxicity
[34]. It has been proposed that GSNO may be an endogenous NO reservoir
that can release NO [35] but also protects against oxidative stress in the
endothelium, myocardium, brain tissue, and other cells [36]. It is possible
then to think that the only decrease or increase in GSH concentration is able
to alter the effects of NO. Another study shows that alterations in GSH levels
change the neurotrophic effects of NO in midbrain cultures into neurotoxic
[37]. Under these conditions, NO triggers a programmed cell death with
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Maria Miranda et al.
markers of both apoptosis and necrosis, the kind of death that is seen in this
retinitis pigmentosa model. Therefore, restoring GSH levels can help NO to
exert its beneficial effects, while the decrease in GSH levels allow more NO
to be free and commit neurotoxic actions.
One of the signaling mechanisms of NO is through the S-nitrosylation of
cysteine residues on proteins. S-nitrosylation is now regarded as an important
redox signaling mechanism in the regulation of different cellular and
physiological functions and deregulation of S-nitrosylation has also been
linked to various human diseases such as neurodegenerative disorders [38].
2.2. Glutathione metabolism in rd1 retina
We have studied several markers of oxidative stress in retinas from rd1
mice and compared them with its values in control mice. MDA, a lipid
peroxidation product, concentration was measured by liquid chromatography
according to a modification of the method of Richard et al [39] as previously
described [40]. There were no differences between MDA values in retina
homogenate from control animals and those from the treated and non treated
rd1 mice (Figure 2a). Glutathione peroxidase (GPx) and glutathione reductase
(GSSG-R) activities were assayed in retina homogenates (Figure 2a and 2b).
GPx activity was assayed as reported by Lawrence et al [41] towards
hydrogen peroxide. GSSG-R activity was assayed as reported by Pinto and
Bartley [42]. GPx is the key enzymatic activity metabolizing cytosolic and
mitochondrial hydrogen peroxide. GPx activity was significantly decreased in
retina from rd1 mice at PN11 compared with controls. There was no
difference in GSSG-R activity between control mice and the rd1 mice retina.
Furthermore, it is also known that there is a reduction in alpha-GST
content in rd1/rd1 retina starting from the second postnatal week and that the
addition of alpha GST exogenously to rd1/rd1 explants was able to rescue
photoreceptor from death [43]. The authors propose that alpha-GST
neuroprotection is mediated by reduction of tissue oxidative stress.
GSH has shown its protective effect on neuronal degeneration by
inhibiting glutamate toxicity [44], a situation also present in retinal
degenerations in the rd1 mouse [45]. Moreover, it has been suggested that
glutathione alterations can also be observed in humans affected with
photoreceptors dystrophy. Two sisters with severe glutathione synthetase
deficiency, an autosomal recessive inborn error of metabolism resulting in
very low intracellular levels of the free-radical scavenger glutathione, showed
progressive retinal dystrophy with hyperpigmentations and maculopathy.
These findings agree with a rod/cone type of retinal dystrophy [46].
Antioxidant therapy in retinitis pigmentosa
7
Figure 2. MDA concentrations, GPx activity and GSSG-R activity, in retinas from
PN 11 control mice, untreated rd1 mice and treated rd1 mice. Values are mean + SD
from at least four animals in each group. (*p<0,05 versus treated, two-tailed Student´s
t-test).
3. Antioxidant treatment in retinitis pifmentosa
Increasing evidence suggests that oxidative stress contributes to the
pathogenesis of many neurodegenerative disorders, including RP, age related
macular degeneration (AMD), glaucoma, diabetic retinopathy, and light
damage [47-49].
8
Maria Miranda et al.
Oxidative damage has also been reported to be present in cone
photoreceptor degeneration [50, 51]. These studies demonstrated the presence
of of acrolein- and 4-hydroxynonenal-adducts on protein; specific indicators
of lipid peroxidation and biomarkers for oxidative damage to proteins and
DNA in experimental animal models of RP at ages in which almost all rods
had died. They postulate the hypothesis that the death of rods results in
decreased oxygen consumption and hyperoxia in the outer retina resulting in
gradual cone cell death from oxidative damage.
In addition, microarray experiments have shown that in the rd1 retina,
genes for products related to protection against oxidative stress are upregulated when compared to wild type mice [52, 53]. Hackman et al [53]
studied gene expression in rd1 retina and compared it with agematched
control retinas at three time points: postnatal day P14, P35, and P50. At each
stage of degeneration, they found there was only limited overlap of the genes
that showed increased expression, suggesting the involvement of temporally
distinct molecular pathways. But they found increased expression of genes
involved in cell proliferation pathways and oxidative stress at each time
point.
In previous studies we have shown that the use of a combination of
antioxidants (zeaxanthin, lutein, α-lipoic acid and glutathione), in vitro and in
vivo drastically reduced the number of rod photoreceptors displaying
oxidatively damaged DNA, and delayed the degeneration process
significantly [54]. For the in vitro studies, antioxidants were added to the
culture medium. For the in vivo studies, postnatal day (PN3) pups of rd1
mice were fed antioxidants either individually or in combination and control
rd1 animals received vehicle alone. The number of TUNEL positive and
avidin positive cells (indicating nuclear oxidative stress) was considerably
decreased upon treatment with the combination of the antioxidants. Rescue of
rd1 photoreceptors was significant at PN18 and PN17, respectively, in the in
vitro and in vivo studies.
Komeima et al have also showed that injecting another combination of
antioxidants (α-tocopherol, ascorbic acid, Mn(III) tetrakis (4-benzoic acid)
porphyrin, and α-lipoic acid) in PN18 rd1 mice decreased cone photoreceptor
cell death [55]. In this study mice were treated with daily injections of the
mixture or each component alone between postnatal day (P)18 and P35.
Between P18 and P35, there was an increase in two biomarkers of oxidative
damage, carbonyl adducts measured by ELISA and immunohistochemical
staining for acrolein, in the retinas of rd1 mice. The staining for acrolein in
remaining cones at P35 was eliminated in antioxidant-treated rd1 mice,
confirming that the treatment markedly reduced oxidative damage in cones;
Antioxidant therapy in retinitis pigmentosa
9
this was accompanied by a 2-fold increase in cone cell density and a 50%
increase in medium-wavelength cone opsin mRNA [55].
These group has also treated with antioxidants animals with other types
of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and
Q344ter mice, a model of rapidly progressive dominant RP [56]. Compared
to appropriate vehicle-treated controls, rd10/rd10 and Q344ter mice treated
between P18 and P35 with a mixture of antioxidants previously found to be
effective in rd1/rd1 mice showed significantly greater cone survival. These
data suggest that oxidative damage contributes to cone cell death regardless
of the disease causing mutation that leads to the demise of rods, and that in
more slowly progressive rod degenerations, oxidative damage may also
contribute to rod cell death [57].
Other studies have also postulated the use of Docosahexaenoic acid
(DHA), the major retinal polyunsaturated fatty acid, to pevent photoreceptor
apoptosis. DHA has long been known to be critical for proper visual function;
its deficiency impairs the electric response to illumination, decreases visual
acuity and affects retinal development [57, 58]. DHA effectively prevents
photoreceptor apoptosis induced by oxidative stress [59] and induces
photoreceptor progenitors to exit the cell cycle [60] and stimulates their
differentiation [61]. Probably DHA prevents photoreceptor apoptosis activating
the ERK/MAPK(ERK)/mitogen-activated protein kinase pathway to promote
photoreceptor survival during early development in vitro and upon oxidative
stress avoiding mitochondrial depolarization [62]. Another possible explanation
for the neuroprotection exerted by DHA, is its transformation in neuroprotectin
Dl (NPDI). NPD1 protects retinal pigment epithelial (RPE) cells from
oxidative stress-induced apoptosis and photoreceptor survival depends on the
integrity of RPE cells [63].
Figure 3. TUNEL staining in retinas from (a) rd1 mice and (b) rd1 mice treated with
antioxidants. Note the decrease in the number of TUNEL positive cells in the ONL
from the treated rd1 mice.
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Maria Miranda et al.
Finally melatonin has also shown to be efective in delaying
photoreceptor loss and reduced the number of apoptotic photoreceptors in the
homozygous rds mouse (rds/rds) [64]. The rds/rds mouse is a spontaneous
mutant, in which photoreceptor cell death is triggered by a null mutation in
the rds/peripherin gene [65]. The mechanisms by which melatonin protects
photoreceptor loss in these animals are unknown.
In view, of the clear alteration of glutathione metabolism, with its
significance in an icrease of oxidative stress, protection from oxidative
damage may be a broadly applicable treatment strategy in RP. Moreover it
has eeen shown that irrespective of whether photoreceptor degeneration is
triggered by gene defects (lack of beta-PDE or rds/peripherin) or
environmental stress (light-damage), a number of pro-apoptotic mechanisms
are triggered leading to the degeneration of the photoreceptor cells [66]. The
temporal pattern of the different pathways suggests that the non-caspasedependent mechanisms may actively participate in the demise of the
photoreceptors. Unless the common upstream initiator for a given
photoreceptor dystrophy is found, multiple rescue paradigms need to be used
to target all active pathways [66]. Since structurally different compounds
with variable antioxidant activity provide additional protection against
increased oxidative stress [67], it appeared reasonable to combine different
antioxidants instead of using them individually, particularly because the
mammalian antioxidant defence system is a complex network and comprise
several enzymatic and non-enzymatic entities [68, 69].
We can then conclude that neuroprotection with antioxidants is a feasible
therapy opportunity to try to maintain photoreceptor survival in the
neuroretina. Anyway, insights into the signalling pathways involved in the
degeneration in this illness are crucial for the development of rational
antioxidant therapies.
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Figure 1 & 3 not mentioned in the text matter. Kindly verify
and confirm.