Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat

Background

Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.

Methods and Findings

We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.

Conclusions

We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.     

Inherited retinal degenerations: therapeutic prospects

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerative diseases, characterized by the progressive death of rod and cone photoreceptors. A tremendous genetic heterogeneity is associated with the RP phenotype. Most mutations affect rods selectively and, through an unknown pathway, cause the rod cells to die by apoptosis. Cones, on the other hand, are seldom directly affected by the identified mutations, and yet, in many cases, they degenerate secondarily to rods, which accounts for loss of central vision and complete blindness. Many animal models of RP are available and have led to a better understanding of the disease and to the development of therapeutic strategies aimed at curing the specific genetic disorder (gene therapy), slowing down or even stopping the process of photoreceptor degeneration (growth factors or calcium blockers applications, vitamin supplementation), preserving the cones implicated in the central visual function (identification of endogenous cone viability factors) or even replacing the lost cells (transplantation, use of stem or precursor cells). Still, many obstacles will need to be overcome before most of these strategies can be applied to humans. In this review, we describe the different therapeutic strategies being studied worldwide and report the latest results in this field.     

Mutations in IMPG1 Cause Vitelliform Macular Dystrophies

Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507^∗). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD.     

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

Background

Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

Methods

To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

Results

The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

Conclusions

In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.     

Neuroprotection of photoreceptor cells in rod-cone dystrophies: from cell therapy to cell signalling

Neuroprotection of photoreceptor cells in rod-cone dystrophies: from cell therapy to cell signalling. Neuroprotection of photoreceptor cells in rod-cone degenerations is primarily targeted at preventing the loss of function. Strategies for protecting rod cells should therefore aim not only at structural preservation but also must be assessed using functional parameters (e.g., electroretinogram). Given the number of mutations leading to an impaired visual response of rods, the preservation of cones is a realistic approach since (1) numerous mutations do not affect proteins expressed by cones; (2) the secondary degeneration of cones is the main event leading to profound visual impairment; (3) even a small proportion of functional cones is sufficient for major visual functions. Our group has (1) established and confirmed the existence of non cell autonomous mechanisms promoting cone cell viability; (2) shown that rod cell protection or replacement provides a mean to extend the survival of cones; (3) demonstrated that rod-cone trophic interactions are mediated by diffusible proteins; (4) identified by expression cloning a protein mediating such interactions: RdCVF (Rod-derived Cone Viability Factor). These studies provide clues for broad neuroprotective therapies of rod-cone dystrophies.     

Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse

We have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches.     

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs^∗57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.