Phillippa Cottrill

Transplantation of Photoreceptors Derived From Human Müller Glia Restore Rod Function in the P23H Rat

Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia‐derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC‐derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a‐wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell‐replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.

Arctic reindeer extend their visual range into the ultraviolet

SUMMARY The Arctic has extreme seasonal changes in light levels and is proportionally UV-rich because of scattering of the shorter wavelengths and their reflection from snow and ice. Here we show that the cornea and lens in Arctic reindeer do not block all UV and that the retina responds electrophysiologically to these wavelengths. Both rod and cone photoreceptors respond to UV at low-intensity stimulation. Retinal RNA extraction and in vitro opsin expression show that the response to UV is not mediated by a specific UV photoreceptor mechanism. Reindeer thus extend their visual range into the short wavelengths characteristic of the winter environment and periods of extended twilight present in spring and autumn. A specific advantage of this short-wavelength vision is the use of potential information caused by differential UV reflections known to occur in both Arctic vegetation and different types of snow. UV is normally highly damaging to the retina, resulting in photoreceptor degeneration. Because such damage appears not to occur in these animals, they may have evolved retinal mechanisms protecting against extreme UV exposure present in the daylight found in the snow-covered late winter environment.

Dominant Cone-Rod Dystrophy: A Mouse Model Generated by Gene Targeting of the GCAP1/Guca1a Gene

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.

Developmental dynamics of cone photoreceptors in the eel

BackgroundMany fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?ResultsThe Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their λmax values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction).ConclusionsLarger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells.

Gene expression and protein distribution of ADAMTSL-4 in human iris, choroid and retina

Background Mutations in ADAMTSL4 have recently been shown to be the major cause of autosomal recessive isolated ectopia lentis (IEL). However, the function and ocular localisation of the protein is yet to be fully established. We therefore aimed to confirm the expression of this gene and protein in normal ocular tissue. Methods Donor ocular tissue was obtained within 48 h post-mortem and iris, choroid and retina were isolated for analysis. Expression of mRNA coding for ADAMTSL4 was examined in four eyes using reverse transcription PCR. Protein coding for this molecule was also investigated in two eyes by western blot analysis. Furthermore, the in situ localisation of ADAMTSL4 was investigated in cryostat sections of whole eyes following immunostaining for this protein and confocal analysis of the stained tissue. Results mRNA and protein coding for ADAMTSL4 were both demonstrated to be expressed in iris and choroidal tissue but were absent from the neural retina. Confocal studies revealed ADAMTS-Like 4 to be present in the ciliary body and ciliary processes and also in the retinal pigment epithelium. Conclusions We have confirmed the gene and protein expression of ADAMTSL4 in human ocular tissue. The pattern of expression may suggest further functions of this gene beyond those suggested by its causative role in IEL.