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1.  Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors 
Cell Death & Disease  2014;5(5):e1234-.
Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD.
PMCID: PMC4047865  PMID: 24853412
TUNEL; PARP; necrosis; retina; cGMP
2.  Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice 
Molecular Vision  2014;20:1527-1544.
The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA’s adoption as an anticonvulsant. This generated speculation for VPA’s potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA’s potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA’s effects on the rate of photoreceptor loss in two strains of mice, Pde6brd1/rd1 and Pde6brd10/rd10.
The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6brd1/rd1 mice. Pde6brd1/rd1 mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6brd10/rd10 mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6brd10/rd10 mice to measure retinal function.
In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9–P21) dramatically slowed the loss of rod photoreceptors in Pde6brd1/rd1 mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6brd10/rd10 mice (P17–P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6brd10/rd10 mice earlier (P9–P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9–P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals.
A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6brd1/rd1 mice (P9–P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA’s efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17–P28) started when the central retina was mature.
PMCID: PMC4225157  PMID: 25489226
3.  Infliximab reduces Zaprinast-induced retinal degeneration in cultures of porcine retina 
cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4 to 5% of retinitis pigmentosa (RP), a rare form of retinal dystrophy. Growing evidence suggests that inflammation is involved in the progression of RP. The aims of this study were to corroborate the presence of high TNFα concentration in the eyes of RP patients and to evaluate whether the blockade of TNFα with Infliximab, a monoclonal anti-TNFα antibody, prevented retinal degeneration induced by PDE6 inhibition in cultures of porcine retina.
Aqueous humor from 30 patients with RP and 13 healthy controls were used to quantify the inflammatory mediators IL-6, TNFα, IL-1β, IL-10 by a multiplex enzyme-linked immunosorbent assay (ELISA) system. Retinal explants from pig were exposed to Zaprinast, a PDE6 inhibitor, for 24 hours in the absence or the presence of Infliximab. Cell death was evaluated by TUNEL assay. The number and distribution of caspase-3 positive cells, indirect poly(ADP)ribose polymerase (PARP) activation and glial fibrillary acidic protein (GFAP) content were visualized by immunolabeling. Antioxidant total capacity, nitrites and thiobarbituric acid reactive substances (TBARS) formation were determined to evaluate antioxidant-oxidant status.
IL-6 and TNFα concentrations were higher in the aqueous humor of RP patients than in controls. Infliximab prevented retinal degeneration, as judging by the reduced presence of TUNEL-positive cells, the reduction of caspase-3 activation and also reduction of glial activation, in an ex vivo model of porcine retina. Additionally, Infliximab partially reduced oxidative stress in retinal explants exposed to Zaprinast.
Inflammatory mediators IL-6 and TNFα were elevated in the aqueous humor of RP patients corroborating previous studies suggesting sustained chronic inflammation. Our study suggests that TNFα is playing an important role in cell death in an ex vivo model of retinal degeneration by activating different cell pathways at different cell layers of the retina that should be further studied.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0172-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4200228  PMID: 25301432
Retinal degeneration; Inflammation; Infliximab; Oxidative stress; TNFα; Poly(ADP-ribose); caspase-3; Retinitis pigmentosa; Photoreceptor death
4.  CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents 
PLoS Medicine  2008;5(2):e39.
In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.
Methods and Findings
We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.
CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.
Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium.
Editors' Summary
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in industrialized countries. The macula is the central region of the retina, the tissue at the back of the eye that detects light and converts it into electrical messages that are sent to the brain. In the commonest form of AMD—“dry” AMD—the light-sensitive cells in the retina (the photoreceptors) gradually die. This degeneration might occur because of damage to the retinal pigment epithelium (RPE). This layer of dark cells lies between the photoreceptors and the choroid, the layer of the eye that contains blood vessels and brings oxygen to the retina. The RPE keeps the retina healthy by transferring the right amount of oxygen and nutrients from the choroid to the retina and by removing worn-out photoreceptor outer segments (the part of the photoreceptor that actually absorbs light) in a process called phagocytosis (engulfment and digestion). In addition to photoreceptor degeneration and RPE shrinkage, a layer of the choroid rich in small blood vessels (the choriocapillaris) also shrinks in dry AMD. For affected individuals, all these changes (which experts describe as retinal atrophy and choroidal involution) mean that the sharp central vision that is needed for reading and driving is destroyed, leaving only dim, burred images or a black hole at the center of the vision.
Why Was This Study Done?
Little is known about the molecular mechanisms that underlie dry AMD and, consequently, there is no cure for it. In this study, the researchers have tested whether a molecule called CD36, which is expressed on the surface of RPE cells, is involved in dry AMD. CD36 is a scavenger receptor—which means it binds many potentially harmful molecules including oxidized fats (which are present in the photoreceptor outer segments) and is involved in their phagocytosis. Phagocytosis itself induces the expression of several proteins in the RPE cells, including COX2, a “proangiogenic” protein that stimulates the growth of blood vessels. Putting this information together, the researchers hypothesized that a defect in CD36 might cause the characteristic retinal atrophy (by preventing the phagocytosis of worn-out photoreceptor outer segments) and choroidal involution (by preventing the induction of COX2 expression and consequently the maintenance of the blood vessels in the choroid) of dry AMD.
What Did the Researchers Do and Find?
The researchers first show that retinal degeneration occurs in rats and mice that express no CD36. This degeneration (which included a reduction in the thickness of the retina, the presence of irregularly shaped photoreceptor outer segments, and the detachment of these structures from the RPE) was seen in old but not young animals. Choroidal involution was also seen in these CD36-deficient animals. This change was present in young mice and rats but increased with age so that by one year old, the choriocapillaris looked moth-eaten. Next, the researchers show that although RPE cells taken from normal animals and grown in dishes were able to make COX2 in response to exposure to purified photoreceptor outer segments, RPE cells from CD36-deficient animals did not. The expression of vascular endothelial growth factor (VEGF; a protein that is needed for normal choroidal development and whose expression is controlled by COX2) showed a similar pattern. Finally, the researchers report that COX2 deficiency in mice caused similar age-dependent choroidal involution and similar effects on VEGF expression in RPE cells as CD36 deficiency.
What Do These Findings Mean?
These findings show that CD36 deficiency leads to progressive, age-related degeneration of photoreceptors and choroidal involution in rats and mice. They also show that CD36 deficiency causes this choroidal involution, the key feature of dry AMD, because it leads to down-regulation of COX2 expression (and subsequently reduced VEGF expression) in the RPE. Researchers now need to find out whether this mechanism for the development of dry AMD holds in people—what happens in animals does not necessarily happen in people. If it does, pharmacological activation of CD36 or restoration of CD36 expression in the RPE might eventually provide a way to treat dry AMD.
Additional Information.
Please access these Web sites via the online version of this summary at
MedlinePlus provides links to information on macular degeneration and an encyclopedia page on macular degeneration (in English and Spanish)
Pages on the US National Institutes of Health NIH SeniorHealth site provides text and spoken information about AMD
The US National Eye Institute and the UK Royal National Institute of Blind People also provide information about AMD
Wikipedia has pages on the retina, photoreceptor cells, retinal pigment epithelium, and choroid (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2245984  PMID: 18288886
5.  Non-Invasive Gene Transfer by Iontophoresis for Therapy of an Inherited Retinal Degeneration 
Experimental eye research  2008;87(3):168-175.
Despite extensive research on many of the genes responsible for inherited retinal degenerations leading to blindness, no effective treatment is currently available for patients affected with these diseases. Among the therapeutic approaches tested on animal models of human retinal degeneration, gene therapy using different types of viral vectors as delivery agents has yielded promising results. We report here our results on a non-invasive, non-viral delivery approach using transscleral iontophoresis for transfer of plasmid DNA into mouse retina. Proof of principle experiments were carried out using plasmid containing GFP cDNA to demonstrate expression of the transferred gene in the retina after single applications of iontophoresis. Various parameters for multiple applications of iontophoresis were optimized to sustain GFP gene expression in mouse photoreceptors. Subsequently, repeated iontophoresis of plasmid containing normal β-phosphodiesterase (β-PDE) cDNA was performed in the rd1 mouse, an animal model of autosomal recessive retinitis pigmentosa caused by a mutant β-PDE gene.
In normal mice, transscleral iontophoresis of the GFP plasmid provided a significant increase in fluorescence of the retina in the treated versus non-treated eyes. In rd1 mice, repeated iontophoresis of β-PDE cDNA plasmid partially rescued photoreceptors morphologically, as observed by microscopy, and functionally, as recorded on ERG measurements, without adverse effects. Therefore, transscleral iontophoresis of plasmid DNA containing therapeutic genes may be an efficient, safe and non-invasive method for the treatment of retinal degenerations.
PMCID: PMC2713253  PMID: 18653181
gene transfer; gene therapy; iontophoresis; retina; retinitis pigmentosa; rd1 mouse
6.  Long-term preservation of cone photoreceptors and visual acuity in rd10 mutant mice exposed to continuous environmental enrichment 
Molecular Vision  2014;20:1545-1556.
In human patients and animal models of retinitis pigmentosa (RP), a gradual loss of rod photoreceptors and decline in scotopic vision are the primary manifestations of the disease. Secondary death of cones and gradual, regressive remodeling of the inner retina follow and progress at different speeds according to the underlying genetic defect. In any case, the final outcome is near-blindness without a conclusive cure yet. We recently reported that environmental enrichment (EE), an experimental manipulation based on exposure to enhanced motor, sensory, and social stimulation, when started at birth, exerts clear beneficial effects on a mouse model of RP, by slowing vision loss. The purpose of this study was to investigate in the same mouse the long-term effects of chronic exposure to an EE and assess the outcome of this manipulation on cone survival, inner retinal preservation, and visual behavior.
Two groups of rd10 mutant mice were maintained in an EE or standard (ST) laboratory conditions up to 1 year of age. Then, retinal preservation was assessed with immunocytochemistry, confocal microscopy examination, cone counts, and electron microscopy of the photoreceptor layer, while visual acuity was tested behaviorally with a Prusky water maze.
rd10 mice are a model of autosomal recessive RP with a typical rod-cone, center to the periphery pattern of photoreceptor degeneration. They carry a mutation of the rod-specific phosphodiesterase gene and undergo rod death that peaks at around P24, while cone electroretinogram (ERG) is extinct by P60. We previously showed that early exposure to an EE efficiently delays photoreceptor degeneration in these mutants, extending the time window of cone viability and cone-mediated vision well beyond the phase of maximum rod death. Here we find that a maintained EE can delay the degeneration of cones even in the long term. Confocal and electron microscopy examination of the retinas of the rd10 EE and ST mice at 1 year of age showed major degeneration of the photoreceptor layer in both experimental groups, with small clusters of photoreceptors persisting in the peripheral retina. These vestigial cells were positive for L and M opsins and cone arrestin and represented the residual population of cones. In the retinas of the EE mice, cones were more numerous and less remodeled than in the ST counterparts, albeit virtually devoid of outer segments, as confirmed with electron microscopy (EM) observations. Cone counting in retinal whole mounts showed that rd10 EE mice at 1 year had almost three times as many surviving cones (34,000±4,000) as the ST control mice (12,700±1,800), t test p=0.003. Accordingly, the rd10 EE mice at 1 year of age were still capable of performing the visual water task in photopic conditions, showing a residual visual acuity of 0.138±0 cycles/degree. This ability was virtually absent in the rd10 ST age-matched mice (0.063±0.014), t test, p=0.029. No major differences were detected in the morphology of the neurons of the inner retina between the two experimental groups.
The approaches used to test the effects of an EE were consistent in showing significantly better preservation of cones and measurable visual acuity in 1-year-old rd10 EE mice. We therefore confirm and extend previous findings that showed an EE is an effective, minimally invasive tool for promoting long-lasting retinal protection in experimental models of RP.
PMCID: PMC4225138  PMID: 25489227
7.  IRetinal Organization in the retinal degeneration 10 (rd10) Mutant Mouse: a Morphological and ERG Study 
Retinal degeneration 10 (rd10) mice are a model of autosomal recessive Retinitis Pigmentosa (RP), identified by Chang et al. in 2002. These mice carry a spontaneous mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because of photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. Aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP.
We analyze the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also study retinal function with the electroretinogram (ERG), recorded between P18 and P30.
We find that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells.
Thus, changes in the rd10 retina are very similar to what previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predict that rd10 mice might become excellent models for rescue approaches.
PMCID: PMC2590657  PMID: 17111372
retinitis pigmentosa; bipolar cell; horizontal cell; confocal microscopy; immunocytochemistry; ERG
8.  Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa 
eLife  2013;2:e00824.
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.
eLife digest
Retinitis pigmentosa is an inherited disorder in which the gradual degeneration of light-sensitive cells in the outer retina, known as photoreceptors, causes a progressive loss of sight. Retinitis pigmentosa can also occur as part of a wider syndrome: patients with Usher syndrome, for example, suffer from early-onset deafness and then develop retinitis pigmentosa later in life. Usher syndrome is caused by mutations in any of more than ten genes, but the most commonly affected is USH2A, which encodes a protein called usherin. Mutations in USH2A can also cause retinitis pigmentosa on its own.
Clinical trials are underway to determine whether it is possible to treat various forms of inherited retinal degeneration using gene therapy. This involves inserting a functional copy of the gene associated with the disease into an inactivated virus, which is then injected into the eye. The virus carries the target gene to the light-sensitive photoreceptor cells where it can replace the faulty gene. This could be particularly useful for conditions such as Usher syndrome, in which the early-onset deafness makes it possible to diagnose retinitis pigmentosa before substantial numbers of photoreceptor cells have been lost.
For gene therapy to become a widely used strategy for the treatment of retinal degenerative disease, identification and functional interrogation of the disease-causing gene/mutations will be critical. This is especially true for large highly polymorphic genes such as USH2A that often have mutations that are difficult to identify by standard sequencing techniques. Likewise, viruses that can carry large amounts of genetic material, or endogenous genome editing approaches, will need to be developed and validated in an efficient patient-specific model system.
Tucker et al. might have found a way to address these problems. In their study, they used skin cells from a retinitis pigmentosa patient with mutations in USH2A to produce induced pluripotent stem cells. These are cells that can be made to develop into a wide variety of mature cell types, depending on the exact conditions in which they are cultured. Tucker et al. used these stem cells to generate photoreceptor precursor cells, which they transplanted into the retinas of immune-suppressed mice. The cells developed into normal-looking photoreceptor cells that expressed photoreceptor-specific proteins.
These results have several implications. First, they support the idea that stem cell-derived retinal photoreceptor cells, generated from patients with unknown mutations, can be used to identify disease-causing genes and to interrogate disease pathophysiology. This will allow for a more rapid development of gene therapy strategies. Second, they demonstrate that USH2A mutations cause retinitis pigmentosa by affecting photoreceptors later in life rather than by altering their development. This suggests that it should, via early intervention, be possible to treat retinitis pigmentosa in adult patients with this form of the disease. Third, the technique could be used to generate animal models in which to study the effects of specific disease-causing mutations on cellular development and function. Finally, this study suggests that skin cells from adults with retinitis pigmentosa could be used to generate immunologically matched photoreceptor cells that can be transplanted back into the same patients to restore their sight. Many questions remain to be answered before this technique can be moved into clinical trials but, in the meantime, it will provide a new tool for research into this major cause of blindness.
PMCID: PMC3755341  PMID: 23991284
next-generation sequencing; retinal degeneration; induced pluripotent stem cells; retinal transplantation; retinal cell differentiation; retinitis pigmentosa; Human; Mouse
9.  Functional Rescue of Degenerating Photoreceptors in Mice Homozygous for a Hypomorphic cGMP Phosphodiesterase 6 b Allele (Pde6bH620Q) 
Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6α and PDE6β) and two regulatory (PDE6γ) subunits. In mice homozygous for a nonsense Pde6brd1 allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6brd1 phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6bH620Q was characterized further.
Photoreceptor degeneration in Pde6bH620Q homozygotes was documented by histochemistry, whereas PDE6β expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca2+ recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6bH620Q homozygotes.
Within 3 weeks of birth, the Pde6bH620Q homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6β expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6bH620Q homozygotes exhibited depressed rod function and elevated outer segment Ca2+. Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors.
Pde6bH620Q homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca2+ to promote photoreceptor death. As degeneration in Pde6bH620Q mutants is slower than in Pde6brd1 mice and can be suppressed by Pde6b transduction, this Pde6bH620Q model may provide an alternate means to explore new treatments of RP.
PMCID: PMC2715364  PMID: 18658088
10.  Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats 
PLoS ONE  2011;6(7):e22181.
Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage—key events in apoptotic cell death—were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.
PMCID: PMC3134478  PMID: 21765948
11.  Genetically Modified Neural Stem Cells for a Local and Sustained Delivery of Neuroprotective Factors to the Dystrophic Mouse Retina 
Stem Cells Translational Medicine  2013;2(12):1001-1010.
In two mouse models of retinitis pigmentosa, intravitreal transplantations of adherently cultivated neural stem cells that had been modified to secrete ciliary neurotrophic factor resulted in a sustained delivery of functionally relevant quantities of the cytokine to the dystrophic retina. These transplantations may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based administration of neurotrophic factors in mouse models of photoreceptor degeneration.
A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6brd1 and Pde6brd10 mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6brd1 mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration.
PMCID: PMC3841080  PMID: 24167317
Neural stem cell; Stem cell transplantation; Retinal photoreceptors; Lentiviral vector; Ciliary neurotrophic factor
12.  Targeted inactivation of synaptic HRG4(UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function 
Experimental eye research  2006;84(3):473-485.
HRG4(UNC119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone-rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANT1, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at ~17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration.
PMCID: PMC1820979  PMID: 17174953
retinal degeneration; knock-out model; transgenic model; photoreceptor; synapse; inner segments; outer segments
13.  Interaction between the Photoreceptor-Specific Tubby-like Protein 1 and the Neuronal-Specific GTPase Dynamin-1 
Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins.
Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1–/– mice at a young age.
Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1–/– mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells.
These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1–/– retina.
PMCID: PMC3021943  PMID: 17525220
14.  Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina 
Journal of neurochemistry  2006;98(6):1732-1745.
Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD+ as its substrate, which, in turn, leads to the depletion of NAD+. In efforts to restore the level of NAD+, depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.
PMCID: PMC1766941  PMID: 16903875
ATP levels; ethidium bromide staining; fluorogold labeling; necrosis; poly(APD-ribose) polymerase; poly(ADP-ribosyl)ation; TUNEL labeling; BZ, benzamide; 3-BZ, 3-amino-benzamide; EtBr, ethidium bromide; FG, fluorogold; GCL, ganglion cell layer; INL, inner nuclear layer; PAR, poly(ADP-ribosyl)ation; PARP-1, poly(ADP-ribose) polymerase-1; PBS, phosphate-buffered saline; PJ-34, N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N, -dimethylamino) acetamide hydrochloride; PT, post-treatment; TUNEL, terminal dUTP nick end labelling; WGS, whole goat serum
15.  Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the β-subunit of transducin with digenic disease 
Molecular Vision  2007;13:287-292.
SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.
270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.
Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the α-, β- and γ-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the β-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.
If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.
PMCID: PMC2633482  PMID: 17356515
16.  AAV-Mediated Lysophosphatidylcholine Acyltransferase 1 (Lpcat1) Gene Replacement Therapy Rescues Retinal Degeneration in rd11 Mice 
The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model.
The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination.
Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice.
The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.
This is a comprehensive morphologic, biochemical, electrophysiologic and behavioral analysis of tyrosine capsid mutant AAV8 (Y733F) or triple mutant AAV2 (Y444, 500, 730F)-mediated photoreceptor rescue in rd11 mice, a naturally occurring retinal degeneration model caused by Lpcat1 mutation.
PMCID: PMC3968931  PMID: 24557352
rd11; gene therapy; mice; Lpcat1; AAV
17.  Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress 
Molecular Vision  2009;15:168-180.
Retinitis pigmentosa (RP), a retinal neurodegenerative disorder characterized by apoptosis of photoreceptor cells, is caused by mutations in many different genes. We analyzed the RP gene ceramide kinase-like (CERKL) to determine CERKL function and contribution to pathogenesis.
RT–PCR was performed to characterize CERKL expression in many human adult and fetal tissues, including retina. We analyzed the protein subcellular localization by confocal microscopy and further verified it by sucrose gradients. We performed lipid kinase activity assays. And finally, we studied the effects on cell apoptosis after CERKL overexpression in transiently transfected cultured cells by propidium iodide staining and poly-(ADP-ribose)-polymerase (PARP) caspase-dependent cleavage.
CERKL transcripts underwent alternative splicing. In the human retina, four different CERKL isoforms of 532, 558, 419, and 463 amino acids were expressed. CERKL proteins were mainly localized in the endoplasmic reticulum and Golgi compartments, but they also shifted localization to nuclei and nucleoli. We also found that CERKL prevented cells from entering apoptosis induced by oxidative-stress conditions.
CERKL remains a unique orphan lipid kinase in that no candidate substrate has been identified after intense research. The dynamic localization of CERKL suggests multiple sites of action. Remarkably, CERKL (but not the RP R257X mutant) exerts a protective role in cells against oxidative stress, consistent with RP mutations impairing the normal protein function in photoreceptors and thus tilting the balance toward apoptosis. These results provide valuable insights into the molecular mechanisms causing retinal degeneration.
PMCID: PMC2628313  PMID: 19158957
18.  Wild type cone photoreceptors persist despite neighboring mutant cone degeneration 
In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant (pde6cw59) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone death in vivo in pde6cw59 fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild type (WT) and pde6cw59 cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones.
PMCID: PMC2805452  PMID: 20053919
Photoreceptors; Bystander Effect; Zebrafish; Apoptosis; Phosphodiesterase; Retinal Degeneration
19.  In Vivo Confocal Intrinsic Optical Signal Identification of Localized Retinal Dysfunction 
The purposes of this study were to investigate the physiological mechanism of stimulus-evoked fast intrinsic optical signals (IOSs) recorded in dynamic confocal imaging of the retina, and to demonstrate the feasibility of in vivo confocal IOS mapping of localized retinal dysfunctions.
A rapid line-scan confocal ophthalmoscope was constructed to achieve in vivo confocal IOS imaging of frog (Rana pipiens) retinas at cellular resolution. In order to investigate the physiological mechanism of confocal IOS, comparative IOS and electroretinography (ERG) measurements were made using normal frog eyes activated by variable-intensity stimuli. A dynamic spatiotemporal filtering algorithm was developed to reject the contamination of hemodynamic changes on fast IOS recording. Laser-injured frog eyes were employed to test the potential of confocal IOS mapping of localized retinal dysfunctions.
Comparative IOS and ERG experiments revealed a close correlation between the confocal IOS and retinal ERG, particularly the ERG a-wave, which has been widely used to evaluate photoreceptor function. IOS imaging of laser-injured frog eyes indicated that the confocal IOS could unambiguously detect localized (30 μm) functional lesions in the retina before a morphological abnormality is detectable.
The confocal IOS predominantly results from retinal photoreceptors, and can be used to map localized photoreceptor lesion in laser-injured frog eyes. We anticipate that confocal IOS imaging can provide applications in early detection of age-related macular degeneration, retinitis pigmentosa, and other retinal diseases that can cause pathological changes in the photoreceptors.
This study investigated the physiological mechanism of stimulus-evoked fast intrinsic optical signal (IOS) recorded in dynamic confocal imaging of the retina, and demonstrated the feasibility of in vivo confocal IOS mapping of localized retinal dysfunctions.
PMCID: PMC3522438  PMID: 23150616
20.  N -Ethyl- N -Nitrosourea Induces Retinal Photoreceptor Damage in Adult Rats 
Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.
PMCID: PMC3320154  PMID: 22481856
N-ethyl-N-nitrosourea; γ-H2A.X; PAR; PARP; photoreceptor cell; retinal degeneration; rat
21.  DHA does not protect ELOVL4 transgenic mice from retinal degeneration 
Molecular Vision  2009;15:1185-1193.
Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3.
Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1–/TG2+, Fat1+/TG2–, and Fat1–/TG2–. All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels.
Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene.
We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.
PMCID: PMC2697457  PMID: 19536303
22.  Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene 
Vision research  2007;47(5):624-633.
We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1 (“r”, “rd”, or “rodless”) mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (β-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for β-PDE immunoreactivity starting at about the same time as wild type (P10), though signal averaged less than 40% of wild type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.
PMCID: PMC2562796  PMID: 17267005
mouse model; retinal degeneration; retinitis pigmentosa; rd1; rd10; nmf137; PDE6b; betaphosphodiesterase; rod photoreceptor; cGMP-PDE; beta-subunit of rod cGMP phosphodiesterase gene
23.  Proinsulin Slows Retinal Degeneration and Vision Loss in the P23H Rat Model of Retinitis Pigmentosa 
Human Gene Therapy  2012;23(12):1290-1300.
Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.
Fernández-Sánchez and colleagues assess the therapeutic potential of adeno-associated virus serotype 1 (AAV1) vector expressing human proinsulin (hPi) on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in a rat model of autosomal-dominant retinitis pigmentosa. hPi expression led to attenuation of visual deterioration, which correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture.
PMCID: PMC3523255  PMID: 23017108
24.  Effects of Combined Ketamine/Xylazine Anesthesia on Light Induced Retinal Degeneration in Rats 
PLoS ONE  2012;7(4):e35687.
To explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats.
Rats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls.
Ketamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p = 0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p = 0.03) were observed in non-treated vs. ketamine-xylazine treated animals.
Our results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.
PMCID: PMC3338443  PMID: 22558200
25.  Ablation of the X-Linked Retinitis Pigmentosa 2 (Rp2) Gene in Mice Results in Opsin Mislocalization and Photoreceptor Degeneration 
Mutations in the RP2 gene are associated with 10% to 15% of X-linked retinitis pigmentosa (XLRP), a debilitating disorder characterized by the degeneration of retinal rod and cone photoreceptors. The molecular mechanism of pathogenesis of photoreceptor degeneration in XLRP-RP2 has not been elucidated, and no treatment is currently available. This study was undertaken to investigate the pathogenesis of RP2-associated retinal degeneration.
We introduced loxP sites that flank exon 2, a mutational hotspot in XLRP-RP2, in the mouse Rp2 gene. We then produced Rp2-null allele using transgenic mice that expressed Cre-recombinase under control of the ubiquitous CAG promoter. Electroretinography (ERG), histology, light microscopy, transmission electron microscopy, and immunofluorescence microscopy were performed to ascertain the effect of ablation of Rp2 on photoreceptor development, function, and protein trafficking.
Although no gross abnormalities were detected in the Rp2null mice, photopic (cone) and scotopic (rod) function as measured by ERG showed a gradual decline starting as early as 1 month of age. We also detected slow progressive degeneration of the photoreceptor membrane discs in the mutant retina. These defects were associated with mislocalization of cone opsins to the nuclear and synaptic layers and reduced rhodopsin content in the outer segment of mutant retina prior to the onset of photoreceptor degeneration.
Our studies suggest that RP2 contributes to the maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in XLRP caused by RP2 mutations. The Rp2null mice should serve as a useful preclinical model for testing gene- and cell-based therapies.
This study reports generation and characterization of a mouse model of Rp2-mediated retinal degeneration and shows that cone opsin mislocalization is an early step in the pathogenesis of associated disease.
PMCID: PMC3700388  PMID: 23745007
Rp2; retina; photoreceptor

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