Pathogenesis Of Retinitis Pigmentosa Research Articles

OR11-002 - Mutations in MVK cause non-syndromic RP

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal disease. Typically beginning with night blindness, RP is characterized by rod cell degeneration followed by cone cell death, which may ultimately lead to complete blindness. Despite extensive knowledge about genes involved in RP pathogenesis, in several cases the genetic cause remains elusive.

Mutations in the Mevalonate Kinase (MVK) Gene Cause Nonsyndromic Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness and peripheral vision loss, and in many cases leads to blindness. Despite extensive knowledge about genes involved in the pathogenesis of RP, the genetic cause remains elusive in many patients. In this study, we aimed to identify novel genes that are involved in the cause of RP. We present a case series with mutations in the mevalonate kinase (MVK) gene. A total of 769 patients with nonsyndromic RP and 174 Dutch control individuals participated in this study. Exome sequencing analysis was performed in a proband of Dutch origin who was initially diagnosed with nonsyndromic autosomal recessive RP. Mutations in MVK were identified and subsequently tested for segregation within the patient's family and screened in a large cohort of patients with genetically unsolved RP. Patients with mutations underwent extensive clinical reexamination. Digital fundus photography, spectral-domain optical coherence tomography (OCT), and fundus autofluorescence analysis were performed in patients with MVK mutations. Mevalonate kinase (MK) enzyme activity was analyzed in cultured lymphoblastoid cells, and mevalonic acid levels were measured in urine samples. Exome variant filtering and prioritization led to the identification of compound heterozygous mutations in MVK (p.I268T and p.A334T) in the proband and her affected brother. Screening of our nonsyndromic RP patient cohort revealed an additional individual who was homozygous for the p.A334T alteration. Clinical reevaluation of all 3 patients showed a classic form of RP with variable extraocular symptoms, such as history of recurrent childhood febrile crises in 2 patients, mild ataxia in 1, and renal failure in 1. All 3 affected individuals showed a significantly decreased MK activity and highly elevated levels of urinary mevalonic acid. Although the MK activity in cells and mevalonic acid concentrations in urine are strongly aberrant and comparable to that in patients with systemic mevalonate kinase deficiency (MKD), only mild clinical symptoms related to this syndrome were observed in our patients. In the current article, we add another phenotype to the spectrum of diverging disorders associated with mutations in MVK.

Relationship between cyclin dependent kinase 5 and apoptosis in the pathogenesis of retinitis pigmentosa in Royal College of Surgeon rats

Objective To observe the expression of cyclin dependent kinase 5 (Cdk5) and p25 in the pathogenesis of retinitis pigmentosa (RP) in Royal College of Surgeon (RCS) rats and its relationships with apoptosis.To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP.Methods Retinas of RCS and RCS-rdy+ rats were obtained at the ages of postnatal day 17,25,35,60.The retinal structure and thickness of outer nuclear layer were measured by optical microscopy.The expression of Cdk5,p25,cleave-caspase 3 in the retina was evaluated by immunohistochemistry.The protein expression of cleave-caspase 3 in the retina was determined by Western blot.The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL).The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software.Results The retinal thickness of the RCS rats was significantly reduced in comparison to the RCS-rdy+ rats as the postnatal days progressed,particularly in the layer of rods and cones and the outer nuclear layer.The expression level of Cdk5,p25,cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days,while decreased on postnatal 60 days; but there was no obvious change of above indexes in RCS-rdy+ rats.The protein expression of cleave-caspase 3 in the RCS rats was significantly increased with progression of postnatal days to postnatal 35; but there was no obvious similar change in RCS-rdy-rats.The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days,while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy+ rats.This study showed that there were significant correlations between the following variables..Cdk5 expression and p25 expression,Cdk5 expression and cleave-caspase 3 expression,Cdk5 expression and apoptotic cells,p25 expression and cleave-caspase 3 expression,p25 expression and apoptotic cells,cleave-caspase 3 expression and apoptotic cells.The partial correlation coefficients were 0.949,0.808,0.959,0.887,0.979,0.852,respectively and the P value was 0.000.Conclusions The apoptotic ceils significantly increases and the expression level of Cdk5,p25,cleavecaspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days.The tendency of apoptotic cells to increase is consistent with the change of Cdk5,p25,cleave-caspase 3 expression.The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats.Cdk5 may be involved in the development of RP in RCS rats. Key words: Retinitis pigmentosa/pathophysiology; Cyclin-dependent kinase 5; Apoptosis; Animal experimentation

Knockout of ccr2 alleviates photoreceptor cell death in a model of retinitis pigmentosa

Neuroinflammation involving CC chemokines such as monocyte chemoattractant protein-1 (MCP-1) has been demonstrated in the pathological process of retinitis pigmentosa (RP), an inherited degenerative retinal disease. However, the mechanism of MCP-1 and its receptor CCR2 involvement in the disease remains unclear. To investigate the role of MCP1/CCR2 in RP pathogenesis, ccr2 mutant RP mice (ccr2−/− rd10) were created and analyzed. The expression of MCP-1, RANTES, stromal cell-derived factor (SDF-1), and tumor necrosis factor-α (TNF-α) in the retinas of wild-type, rd10, and ccr2−/− rd10 mice was analyzed using quantitative RT-PCR. Photoreceptor apoptosis (TUNEL staining) and the number of microglia (positive for the F4/80 antibody) in the retina were examined. Retinal function was assessed using electroretinograms, and the structure of the whole retina was analyzed from images obtained using optical coherence tomography (OCT) and by histological examination. The expression levels of MCP-1, RANTES, and SDF-1 increased with time in the rd10 mice but not in the wild-type mice. Rearing the mice in the dark prevented degeneration and resulted in thicker photoreceptor layers at each time point. In those mice, the peaks of chemokine expression shifted to a later time with degeneration, suggesting that the expression of these chemokines was induced during the progression of degeneration. Although the difference was not so obvious, the retina in the ccr2−/− rd10 mice was consistently and significantly thicker than that in the rd10 (ccr2+/+ rd10) mice at all time points. Rhodopsin gene expression was also higher in the ccr2−/− rd10 mice than in rd10 (ccr2+/+ rd10) mice, suggesting photoreceptor survival in the former. Retinal function was also better preserved in the ccr2−/− rd10 mice than in the rd10 mice. The number of microglia in the retinas of the ccr2−/− rd10 mice was significantly lower than that in the retinas of the rd10 mice. Interestingly, the MCP-1 induction that was observed in the retinas of the rd10 mice was diminished in the retinas of the ccr2−/− rd10 mice. Our results suggest that the MCP-1/CCR2 system plays a role in retinal degeneration in rd mouse retinas. Retinal MCP-1 expression in the rd mouse retina may be partially controlled by ccr2-positive circulating cells.

Clinical Evidence of Sustained Chronic Inflammatory Reaction in Retinitis Pigmentosa

To study the nature of inflammatory reaction in eyes of patients with retinitis pigmentosa (RP) and its possible role in the pathogenesis of RP. Retrospective, observational study. Three hundred seventy-one consecutive patients diagnosed with typical RP were included in this study. We included 165 patients without active inflammatory diseases, including 20 patients diagnosed with cataract, and 36 patients diagnosed with idiopathic epiretinal membrane as controls. Density of the inflammatory cells in the anterior vitreous cavity was measured and graded by slit-lamp biomicroscopy. A multiplex enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the concentration of cytokines and chemokines in aqueous humor and vitreous fluid of patients with RP and controls. In addition, we investigated the relationship between visual function and anterior vitreous cells in these patients. Slit-lamp biomicroscopic analysis, best-corrected visual acuity, visual field analysis, and multiplex ELISA. In 190 of 509 eyes with RP (37.3%), "1+" (5-9 cells per field) or more cells were observed in the anterior vitreous cavity. Strong inflammatory reaction with "2+" cells (10-30 cells per field) was associated with younger age. In the elderly patients with RP, significantly decreased visual function was seen in a group with "1+" or more cells (P<0.05). Moreover, the levels of a variety of proinflammatory cytokines and chemokines, including monocyte chemotactic protein-1, were increased both in the aqueous humor and vitreous fluid of RP patients compared with the levels in control patients. Sustained chronic inflammatory reaction may underlie the pathogenesis of RP, suggesting interventions for ocular inflammatory reaction as a potential treatment for patients with RP. The authors have no proprietary or commercial interest in any of the materials discussed in this article.

MutT Homolog-1 Attenuates Oxidative DNA Damage and Delays Photoreceptor Cell Death in Inherited Retinal Degeneration

Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP.

Low levels of plasma endothelin-1 in patients with retinitis pigmentosa

The aim of this study was to elucidate the role of endothelin-1 (ET-1) in the pathophysiology of retinitis pigmentosa (RP). Plasma ET-1 levels and ophthalmic features in 50 RP patients were compared with those in 20 healthy-eye control subjects. Plasma ET-1 concentrations were determined using a commercially available enzyme-linked immunosorbent assay kit. Mean plasma ET-1 levels of RP patients (1.88 +/- 0.56 pg/mL) were significantly lower than those of control subjects (2.30 +/- 0.30 pg/mL, Mann-Whitney's U test; P < 0.01). However, ET-1 concentrations varied markedly in each patient. Among RP patients, a significant correlation of ET-1 concentrations was not observed in terms of its hereditary forms or other clinical factors. ET-1 may be important in the pathogenesis of RP, and measurement of its plasma concentrations may also contribute to additional insights into the retinal hemodynamics of RP.

Lipofuscin- and melanin-related fundus autofluorescence visualize different retinal pigment epithelial alterations in patients with retinitis pigmentosa

To compare melanin-related near-infrared fundus autofluorescence (FAF; NIA, excitation 787 nm, emission >800 nm) with lipofuscin-related FAF (excitation 488 nm, emission >500 nm) in retinitis pigmentosa (RP). Thirty-three consecutive RP patients with different modes of inheritance were diagnosed clinically, with full-field ERG, and if possible with molecular genetic methods. FAF and NIA imaging were performed with a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph 2). Rings of increased FAF were present within an area of preserved retinal pigment epithelium (RPE) at the posterior pole (31/33). Rings of increased NIA were located in the same region as rings of increased FAF. In contrast to FAF, NIA showed a precipitous decline of NIA peripheral to the ring. In larger areas of preserved NIA (11/31), pericentral and foveal NIA were of similar intensity with an area of lower NIA in between. In smaller areas of preserved NIA (20/31), NIA was homogeneous from the perifovea to the fovea. In one patient without a ring of increased FAF, NIA distribution was normal. In the remaining patient with severely advanced RP, no residual RPE as well as no FAF and NIA were detectable. Characteristic features for FAF and NIA alterations in a heterogeneous group of RP patients indicate a common pathway of RPE degeneration. Patterns of NIA and FAF indicate different pathophysiologic processes involving melanin and lipofuscin. Combined NIA and FAF imaging will provide further insight into the pathogenesis of RP and non-invasive monitoring of future therapeutic interventions.

R659S mutation of γPKC is susceptible to cell death: Implication of this mutation/polymorphism in the pathogenesis of retinitis pigmentosa

It has been reported that mutations of γPKC cause hereditary spinocerebellar atrophy type 14 (SCA14). Our recent study has revealed that the SCA14 mutant γPKC is susceptible to aggregation and causes cell death. Among mutations/polymorphisms of γPKC, the R659S mutation was firstly segregated from families with hereditary retinitis pigmentosa type 11 (RP11). Although more reliable etiological mutations of RP11 were subsequently discovered in a human homologue of yeast pre-mRNA splicing gene (PRP31), the role of this R659S missense change in the pathogenicity of RP11 is still controversial. In this study, we overexpressed R659S γPKC in CHO cells and characterized the properties of this mutant protein. We found that R659S γPKC more prominently induced cell death than did wild-type. This mutant γPKC had higher basal activity than wild-type, however, no difference was found in the extent of aggregation and insolubility to detergent between R659S mutant and wild-type. These results suggest that the R659S mutation is susceptible to neuronal death and is involved in the pathogenesis of neurodegenerative diseases, including RP11.

Digenic association of RHO and RP1 genes with retinitis pigmentosa among Chinese population in Hong Kong

To identify the mutation patterns of RHO and RP1 genes in the Chinese patients with retinitis pigmentosa (RP) and to explore their potential interactions in the pathogenesis of RP. Sequence alterations in the entire coding region and splice sites of RHO and RP1 gene were screened in 151 RP affected probands and 150 unrelated controls who were all Hong Kong Chinese. Additional 46 relatives of 12 RP probands carrying possible mutations in RHO or RP1 were recruited for segregation analysis. Univariate analysis, multivariate analysis and genotype-pedigree disequilibrium test were used to examine the associations of polymorphisms in these two genes with RP. Two mutations in the RHO gene, 5211delC and P347L, were identified each in one proband from the 151 probands, accounting for 1.3% of the RP patients. Two mutations in the RP1 gene, R677X and D984G, were identified each in one proband from the 151 probands, also accounting for 1.3% of the RP patients. In univariate analysis, non-coding sequence variants in the RHO gene, -26G > A, was found to increase the risk of RP, while R872H in the RP1 gene was likely to be a protective factor for RP. Multivariable logistic regression analysis and haplotype analysis confirmed these associations. The prevalences of RHO and RP1 mutations among the RP patients in Chinese population are both less than reported in other populations. Besides the disease-causing mutations, non-coding sequence alterations may also be a modifier for RP. The potential interactions between RHO and RP1 suggest a digenic etiology for RP.

Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.

POLYMORPHISMS OF DOPAMINE RECEPTORS IN PATIENTS WITH RETINITIS PIGMENTOSA

Background. Dopamine (DA) has a specific role in modulation of retinal function, renewal and phagocytosis of shed discs by the retinal pigment epithelium. Animal model of RCS (Royal College of Surgeons) rats which have impaired retinal phagocytosis has shown an appearance similar to the clinical picture seen in patients with advanced retinitis pigmentosa (RP). Based on RCS rats’ studies and the fact that DA has an important role in retinal renewal we assume that certain DA receptor polymorphisms might play a role in pathogenesis of RP. Materials and methods. We compared a group of 65 RP patients and 80 healthy individuals. Using PCR method and restriction with DdeI, TaqI or MspI restriction enzymes (DRD1, DRD2, DRD3 respectively) we determined the polymorphisms of DRD1, DRD2 and DRD3. Three models of expression (codominant, dominant, recessive) were statistically compared with χ 2-test. Results. We found an evidence for association between DRD2 TaqI RFLP, OR = 1.9 (95% CI: 1.7–2.3), p = 0.08, under autosome recessive model of inheritance. Other models for any of the DRD polymorphisms did not show a significant association with RP. Conclusions. A potential association was found between RP and DRD2 polymorphism. Further investigation is needed to confirm potential implication of DRD2 in the pathogenesis of RP.

Gene therapy for ocular disease.

Gene therapy for ocular disease.

Investigating retinitis pigmentosa: a laboratory scientist's perspective

Abstract A decade's search for the molecular basis of retinitis pigmentosa (RP) has uncovered an unexpected genetic heterogeneity. These experimental results raise again the fundamental question concerning the pathogenesis of RP: mutations in a number of different genes all result in a similar phenotype of the disease—the gradual loss of photoreceptors. At the very least, this genetic heterogeneity allows us to make one important inference: it is cellular mechanisms other than the genetic defect alone which bring on the symptoms and clinical course typical of RP. Furthermore, once they have begun to express themselves, this multitude of RP-inducing mutations seem to converge into a common set of cellular mechanisms that eventually lead to photoreceptor death. In those cases where the RP-inducing mutation is in a rod photoreceptor-specific gene, cell-cell interactions may lead to the death of normal cone photoreceptors. RP researchers are now confronted by three related challenges: to pinpoint the immediate physiologic effects of the various RP-inducing mutations; to identify the mechanisms directly causing photoreceptor death; and, to delineate the link between a specific mutation and the direct cause of photoreceptor death.

Red blood cell membrane phosphatidylethanolamine fatty acid content in various forms of retinitis pigmentosa

In order to test the hypothesis that retinitis pigmentosa (RP) is associated with fatty acid abnormalities within cell membrane phospholipids, red blood cell membrane (RBC) phosphatidylethanolamine (PE) fatty acid content (% of total fatty acids) was measured using high performance liquid chromatography and capillary column gas chromatography in 155 patients from separate families with different genetic types of RP and 101 normal subjects. After controlling for the effects of age and sex, patients with all genetic forms of RP had significantly (P < 0.001) reduced mean RBC PE 22:6 omega 3 (n-3) (docosahexaenoic acid, DHA) content, and significantly (P < 0.001) elevated mean RBC PE dimethyl acetal (DMA) forms of 16:0, 18:0, and 18:1 omega 9 (n-9) as compared with normal subjects. RBC PE content of 22:5 omega 3 (n-3) (a precursor to DHA) and 18:2 omega 6 (n-6) (the major dietary essential fatty acid) were not significantly different in RP than in controls. Analysis by genetic types of RP showed that the mean RBC PE DHA percentages were significantly reduced by 24%, 14%, 30%, and 17%, respectively, in dominant, recessive, X-linked, and isolate forms of RP. The relative amounts of plasmalogens as indicated by DMA forms of 16:0 and 18:0 were significantly (P < 0.01) increased in dominant (by 33% and 25%), recessive (by 36% and 25%), and isolate cases (by 32% and 26%) of RP as compared with normal subjects. No such differences were seen in X-linked cases versus controls. Our data indicate that RBC PE DHA content is decreased in all genetic types of RP patients as compared to control subjects, and that RBC PE plasmalogens are increased in dominant, recessive, and isolate forms of RP. These data raise the possibility that membrane phospholipid fatty acid abnormalities may contribute to the pathogenesis of RP.

Immune alterations associated with T lymphocyte activation and regulation in retinitis pigmentosa patients

Altered immunoregulation, suggested previously in the heredo-familial retinal degeneration, retinitis pigmentosa (RP), led us to examine cell-mediated immune responses in 58 RP patients who expressed either recessive or dominant inheritance. Increased absolute numbers of activated T-cells, quantitated by flow cytometry using the Tal epitope, were found in the peripheral blood of 60% of these RP patients. The unusual finding was equally distributed between dominant (16 of 25) and recessive (19 of 33) types of RP, suggesting an immune process present in both patterns of heredity. Additional altered lymphocyte immunoregulation was suggested in all RP by lymphocyte responses to stimulants modified by indomethacin or histamine in vitro. Although no clear association could be found between Tal expression and demographic factors including age, sex, years with RP symptoms, or percentage of life with RP disease, the significantly altered immunoregulatory responses in RP may be related to the pathogenesis of RP.

Light deprivation and retinitis pigmentosa

The historical development of the idea that light deprivation could have therapeutic benefit for patients with retinitis pigmentosa is reviewed. A patient with autosomal recessive and a patient with autosomal dominant retinitis pigmentosa participated in a trial of light deprivation of one eye with an opaque scleral contact lens for 5 yr for about 6–8 hr per day. Each showed comparable progression in both eyes as monitored by changes in visual acuity, visual fields, fundus appearance, and electroretinograms (ERGs). In addition to the well known abnormality in dark adaptation with consequent night blindness, studies of cone ERG b-wave implicit times have shown that patients with early retinitis pigmentosa can have a defect in the mechanism by which their cone system adapts to light. Until more is known about this defect and the pathogenesis of retinitis pigmentosa, we recommend that patients wear dark sunglasses for outdoor use although we have no evidence at this time to substantiate that light deprivation with any type of sunglass or use of an opaque lens will modify the rates of progression of these diseases.

LAURENCE-MOON-BIEDL SYNDROME

In recent years, with the advent of newer knowledge concerning vitamins, hormones and the autonomic nervous system, many clinicians have shown renewed interest in the problem of the pathogenesis of retinitis pigmentosa. As a result patients with this disease have been gorged with vitamins, plied with hormones and subjected to cervical sympathectomy in an effort to arrest or cure the condition. Without wishing to be therapeutic nihilists, we believe that these efforts have been misguided. This belief has been reenforced by a study of the Laurence-Moon-Biedl syndrome in 2 instances. In view of the fact that classic examples of the Laurence-Moon-Biedl syndrome are so rare as to be of great interest and are full of significant implications with regard to the polyglandular and retinal manifestations, it is felt that these cases are worthy of report. REPORT OF CASES Case1.—A. Z., a 12 year old white Cuban boy, was referred

PATHOGENESIS OF RETINITIS PIGMENTOSA

In the past, studies on the pathogenesis of retinitis pigmentosa have been concerned mainly with efforts to discover the remote causes of the disease : the influence of heredity and of consanguinity between the parents, the possible disturbance of liver function, the possible relation of vitamin deficiency, the relation of the ocular disturbance to associated conditions in other organs such as deafness, endocrine disturbances, mental defect, epilepsy, etc. The aim of the present investigation is much more modest, and is concerned with an attempt to unravel the local manifestations of the disease, and to discover what aspect of the local lesion may be considered primary and what aspects may be considered secondary to this primary lesion. For example, the disease process manifests itself in the death and destruction of the retinal neuro-epithelium, and of the retinal ganglion cells. One of the questions that we wish to