Björn Ekesten

Inactivation of Pmel Alters Melanosome Shape But Has Only a Subtle Effect on Visible Pigmentation

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel −/−). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel −/− melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

Cone and rod inputs to murine retinal ganglion cells: evidence of cone opsin specific channels.

To identify ultraviolet (UV) and middle- (M) wavelength-sensitive cone and rod signals in murine retinal ganglion cells, single ganglion cell responses were studied in anesthetized, light-adapted C57/BL6 mice with tungsten microelectrodes driven through the sclera and vitreous to the neural retina. One hundred fifty-four ganglion cells were examined in 43 retinas of 34 mice. The retina was stimulated with diffuse flashes and/or pulses of ultraviolet (360 nm) or green (520 nm) light in the presence and absence of a strong steady orange adapting light. Twelve ganglion cells were studied in the dark-adapted retina in order to identify the signals of rods. Three functionally different types of ganglion cells were found: (1) phasic responding cells (31%) with no spontaneous activity and large impulse amplitudes; (2) tonic responding cells (60%) with irregular, low frequency (5-10 Hz) spontaneous activity and smaller impulse amplitudes; and (3) metronome-like cells (9%) with regular, relatively high-frequency (20-40 Hz) spontaneous activity. A few cells (1%) had habituating responses. Every cell encountered was affected by diffuse stimulation. The more common two types were excited at either the ON or OFF or at both the ON and OFF phases of stimulation. Type III cells had weaker responses, sometimes only inhibited by turning off a light. In the light-adapted state, most cells received signals of the same polarity from UV- and M-cones but UV-cone inputs were usually more dominant, especially in ventral retina. A fraction of cells received signals from only UV- (18%) or only M- (3%) cones. In rare cases (2%) these cone inputs had an opposite polarity on the same cell. In the dark-adapted state, all cells were at least four or five logarithmic units more sensitive and more to green than ultraviolet light. The results indicate that co-expression of both UV-and M-cone opsins cannot be ubiquitous in murine retina. Some cones, especially UV cones, exist without the presence of any functional M-cone opsin. This must be the case to explain the presence of ganglion cells that receive inputs only from UV-cones and others that receive inputs of opposite polarity from UV- and M-cones. The results support the hypothesis that murine retina has the physiological capacity to relay signals to the brain that allow the sensing of chromatic contrast and color vision.

Cone inputs to murine retinal ganglion cells.

Responses of single retinal ganglion cells in different areas of mouse retina were studied to determine their cone inputs, using spectral sensitivity functions and chromatic adaptation. Spectral sensitivity curves were based on threshold response criteria to full field stimulation. The retina of the mouse was viewed through a dilated pupil with a surgical microscope. Ganglion cells were classified into three groups: one receiving inputs from short wave sensitive cones, a second receiving inputs from only middle wavelength sensitive cones and a third receiving inputs from both of these types of cones. The ventral retina, contained a large fraction of the first group of ganglion cells. The dorsal retina and the border between these two areas contained relatively more of the latter two groups. A small fraction of cells were found which displayed antagonistic-like interactions between photoreceptor systems. The results demonstrate that single ganglion cells in mouse retina can select responses from only one of the two cone mechanisms present in this retina, even in areas containing both types of cones.

Multiple congenital ocular anomalies in Icelandic horses

BackgroundMultiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.ResultsWe examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies.One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.ConclusionsThe MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.

Cone properties of the light-adapted murine ERG

Purpose: Because the mouse lacks a typical Purkinje shift, we have examined its light-adapted ERG to determine whether there was other evidence in addition to tolerance to background light, that could be used to identify cone function in the ERG. Methods: Full field comeal ERGs to white flashes, double flashes and flash trains were examined in the presence of a strong full field light adaptation and compared with the human cone ERG. Results: The following cone-like properties could be identified. (1) The light-adapted murine ERG increases in amplitude gradually during the first 10 minutes of light-adaptation; (2) It is capable of responding to a 50 Hz stimulus, although its overall frequency response is slower than that of the human cone ERG; (3) A corneal positive d-wave occurs to the termination of a flash train; (4) The response increases linearly with light intensity. Conclusion: The light-adapted murine ERG has several properties of cones but it has a slower response than the human cone ERG.

Ultraviolet and middle wavelength sensitive cone responses in the electroretinogram (ERG) of normal and Rpe65 −/− mice

Ultra-violet (UV) and middle wavelength sensitive (M) cone responses were identified in the ERG of normal and Rpe65 -/- mice using chromatic flashes and selective chromatic adaptation. In normal mice, the UV-cone response was as large as, or larger, in the presence of a bright yellow adapting light than it is in the presence of a dim white light. The M-cone response became undetectable in the presence of the yellow adapting light. Yellow adapting light initially reduced the UV response, but it recovered in 8-10 min. The M-cone response did not recover. UV-cone responses were undetectable in Rpe65 -/- mice. The M-cone response of young Rpe65 -/- mice was almost as large as in normal mice. A yellow adapting light only diminished this M-cone response. With age, the M-cone response further decreased in Rpe -/- mice. We show a pronounced loss of UV-cone function in Rpe65 -/- mice, which may be related to a defect UV-cones share with rods. The M-cone function is also affected already in young Rpe65 -/- mice. The transient effect of a yellow adapting light on the UV-cone response of normal mice is suggested to be neural, because it disappears during maintained light adaptation.

Equine Multiple Congenital Ocular Anomalies and Silver Coat Colour Result from the Pleiotropic Effects of Mutant PMEL

Equine Multiple Congenital Ocular Anomalies (MCOA) syndrome is a heritable eye disorder mainly affecting silver colored horses. Clinically, the disease manifests in two distinct classes depending on the horse genotype. Horses homozygous for the mutant allele present with a wide range of ocular defects, such as iris stromal hypoplasia, abnormal pectinate ligaments, megaloglobus, iridociliary cysts and cataracts. The phenotype of heterozygous horses is less severe and predominantly includes iridociliary cysts, which occasionally extend into the temporal retina. In order to determine the genetic cause of MCOA syndrome we sequenced the entire previously characterized 208 kilobase region on chromosome 6 in ten individuals; five MCOA affected horses from three different breeds, one horse with the intermediate Cyst phenotype and four unaffected controls from two different breeds. This was performed using Illumina TruSeq technology with paired-end reads. Through the systematic exclusion of all polymorphisms barring two SNPs in PMEL, a missense mutation previously reported to be associated with the silver coat colour and a non-conserved intronic SNP, we establish that this gene is responsible for MCOA syndrome. Our finding, together with recent advances that show aberrant protein function due to the coding mutation, suggests that the missense mutation is causative and has pleiotrophic effect, causing both the horse silver coat color and MCOA syndrome.

Corneal cross-linking in 9 horses with ulcerative keratitis

BackgroundCorneal ulcers are one of the most common eye problems in the horse and can cause varying degrees of visual impairment. Secondary infection and protease activity causing melting of the corneal stroma are always concerns in patients with corneal ulcers. Corneal collagen cross-linking (CXL), induced by illumination of the corneal stroma with ultraviolet light (UVA) after instillation of riboflavin (vitamin B2) eye drops, introduces crosslinks which stabilize melting corneas, and has been used to successfully treat infectious ulcerative keratitis in human patients. Therefore we decided to study if CXL can be performed in sedated, standing horses with ulcerative keratitis with or without stromal melting.ResultsNine horses, aged 1 month to 16 years (median 5 years) were treated with a combination of CXL and medical therapy. Two horses were diagnosed with mycotic, 5 with bacterial and 2 with aseptic ulcerative keratitis. A modified Dresden-protocol for CXL could readily be performed in all 9 horses after sedation. Stromal melting, diagnosed in 4 horses, stopped within 24 h. Eight of nine eyes became fluorescein negative in 13.5 days (median time; range 4–26 days) days after CXL. One horse developed a bacterial conjunctivitis the day after CXL, which was successfully treated with topical antibiotics. One horse with fungal ulcerative keratitis and severe uveitis was enucleated 4 days after treatment due to panophthalmitis.ConclusionsCXL can be performed in standing, sedated horses. We did not observe any deleterious effects attributed to riboflavin or UVA irradiation per se during the follow-up, neither in horses with infectious nor aseptic ulcerative keratitis. These data support that CXL can be performed in the standing horse, but further studies are required to compare CXL to conventional medical treatment in equine keratitis and to optimize the CXL protocol in this species.

Cone inputs to murine striate cortex

BackgroundWe have recorded responses from single neurons in murine visual cortex to determine the effectiveness of the input from the two murine cone photoreceptor mechanisms and whether there is any unique selectivity for cone inputs at this higher region of the visual system that would support the possibility of colour vision in mice. Each eye was stimulated by diffuse light, either 370 (strong stimulus for the ultra-violet (UV) cone opsin) or 505 nm (exclusively stimulating the middle wavelength sensitive (M) cone opsin), obtained from light emitting diodes (LEDs) in the presence of a strong adapting light that suppressed the responses of rods.ResultsSingle cells responded to these diffuse stimuli in all areas of striate cortex. Two types of responsive cells were encountered. One type (135/323 – 42%) had little to no spontaneous activity and responded at either the on and/or the off phase of the light stimulus with a few impulses often of relatively large amplitude. A second type (166/323 – 51%) had spontaneous activity and responded tonically to light stimuli with impulses often of small amplitude. Most of the cells responded similarly to both spectral stimuli. A few (18/323 – 6%) responded strongly or exclusively to one or the other spectral stimulus and rarely in a spectrally opponent manner.ConclusionMost cells in murine striate cortex receive excitatory inputs from both UV- and M-cones. A small fraction shows either strong selectivity for one or the other cone mechanism and occasionally cone opponent responses. Cells that could underlie chromatic contrast detection are present but extremely rare in murine striate cortex.

A slowly progressive retinopathy in the Shetland Sheepdog.

OBJECTIVE To describe a slowly progressive retinopathy (SPR) in Shetland Sheepdogs. Animals  Forty adult Shetlands Sheepdogs with ophthalmoscopic signs of SPR and six normal Shetland Sheepdogs were included in the study. PROCEDURE Ophthalmic examination including slit-lamp biomicroscopy and ophthalmoscopy was performed in all dogs. Electroretinograms and obstacle course-test were performed in 13 affected and 6 normal dogs. The SPR dogs were subdivided into two groups according to their dark-adapted b-wave amplitudes. SPR1-dogs had ophthalmoscopic signs of SPR, but normal dark-adapted b-wave amplitudes. Dogs with both ophthalmoscopic signs and subnormal, dark-adapted b-wave amplitudes were assigned to group SPR2. Eyes from two SPR2 dogs were obtained for microscopic examination. RESULTS The ophthalmoscopic changes included bilateral, symmetrical, greyish discoloration in the peripheral tapetal fundus with normal or marginally attenuated vessels. Repeated examination showed that the ophthalmoscopic changes slowly spread across the central parts of the tapetal fundus, but did not progress to obvious neuroretinal thinning presenting as tapetal hyper-reflectivity. The dogs did not appear seriously visually impaired. SPR2 showed significantly reduced b-wave amplitudes throughout dark-adaptation. Microscopy showed thinning of the outer nuclear layer and abnormal appearance of rod and cone outer segments. Testing for the progressive rod-cone degeneration ( prcd )-mutation in three dogs with SPR was negative. CONCLUSION Slowly progressive retinopathy is a generalized rod-cone degeneration that on ophthalmoscopy looks similar to early stages of progressive retinal atrophy. The ophthalmoscopic findings are slowly progressive without tapetal hyper-reflectivity. Visual impairment is not obvious and the electroretinogram is more subtly altered than in progressive retinal atrophy. The etiology remains unclear. SPR is not caused by the prcd-mutation.