Reductions in Taurine Secondary to Photoreceptor Loss in Irish Setters with Rod-Cone Dysplasia

Transcription

1 Reductions in Taurine Secondary to Photoreceptor Loss in Irish Setters with Rod-Cone Dysplasia S. Y. Schmidr*t and G. D. Aguirre$ These studies show that onset of photoreceptor cell degeneration preceded the loss of taurine in retinas of Irish setters with rod-cone dysplasia. The numbers of photoreceptor cell nuclei were within the normal adult range in affected setters at 10 through 26 days of age but declined rapidly between 26 and 45 days and more gradually thereafter; their numbers became reduced to 50% of normal at 45 days and then to 12-20% and 3-10% of normal at 192 and 346 days, respectively. Taurine concentrations increased within the photoreceptor cell layer during normal development to peak values (50 mm) at a time (45 days of age) corresponding to the development of adult photoreceptor function. In the affected setters, taurine levels increased as in the normal until 26 days of age and then remained at that value until 40-50% of the photoreceptor cells had degenerated. Thereafter, taurine levels declined gradually throughout the period of photoreceptor cell degeneration and were reduced to 30-40% of the normal adult level at the time (346 days) when the thickness of the outer nuclear layer was reduced to less than one complete row of nuclei. These observations agree with findings in retinal degeneration (rd) mice and RCS rats and indicate that in all three of these animal models of hereditary retinal degenerations, reductions in retinal taurine levels occur secondary to the loss of photoreceptor cells. Invest Ophthalmol Vis Sci 26: , 1985 Taurine, the predominant free amino acid within the retina, has been shown to be concentrated within the photoreceptor cell layer in all vertebrates so far studied. 1 " 3 Evidence has indicated that taurine has an essential role in maintaining the normal structure and function of the photoreceptor cells; cats deprived of dietary taurine were shown to develop retinal taurine deficiency associated initially with photoreceptor cell malfunction and subsequently with photoreceptor degeneration. 45 Although these studies have provided evidence that taurine is important for photoreceptor cell viability in a model of nutritionally induced photoreceptor cell degeneration, it was not clear whether or not taurine deficiency played a role in a hereditary photoreceptor cell degeneration as occurs in Irish setters with rod-cone dysplasia. From the Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary,* Boston, Massachusetts, and Sections of Medical Genetics, School of Veterinary Medicine and the Scheie Eye Institute, Department of Ophthalmology, School of Medicine, University of Pennsylvania,! Philadelphia, Pennsylvania. Supported by Research Grants EY-01687f and EY from the National Eye Institute and by Grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, the Elaine O. Weiner Teaching and Research Fund, and the American Irish Setter Foundation. Submitted for publication: August 28, Reprint requests: S. Y. Schmidt, PhD, Berman-Gund Laboratory, Harvard Medical School, 243 Charles Street, Boston, MA Different patterns of developmental changes in retinal taurine concentrations have been previously noted in two animal models with hereditary retinal degeneration: The levels of taurine failed to show the normal increase during photoreceptor cell development in retinal degeneration mutant (rd) mice, 6 while they reached normal adult levels in retinas of Royal College of Surgeons (RCS) rats. 7 The hereditary abnormality in Irish setters seemed similar to that in rd mice, since in both species a failure of normal outer segment development and a deficiency in the activity of a photoreceptor cyclic GMP phosphodiesterase (PDE) has been noted. 8 " 12 In contrast, in RCS rats, normal photoreceptor cell development and normal postnatal increases in PDE activity have been reported The present studies were done to evaluate changes in taurine concentrations within retina and microdissected photoreceptor cell layer in relation to the time course of photoreceptor cell development and photoreceptor cell death in Irish setters with rod-cone dysplasia. For comparison, taurine concentrations within the retina were also measured at defined stages of photoreceptor cell development and degeneration in rd mice and RCS rats. Materials and Methods Irish setters homozygous for the rod-cone dysplasia gene, and control setters (phenotypically normal het- 679

2 680 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Moy 1985 Vol. 26 erozygotes or normal controls) were obtained from a colony (Federated Animal Resources; Honeybrook, PA) supported in part by the National Eye Institute/ National Institutes of Health and in part by the National Retinitis Pigmentosa Foundation. The animals were raised in indoor/outdoor kennels under ambient illumination. Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School, the ARVO Resolution on the Use of Animals in Research, and the guidelines prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council [DHEW (DHHS) publication No. (NIH) 78-23, revised 1978]. The dogs were killed at various ages between 10 and 365 days of age. Prior to analysis of retinal taurine concentrations, dogs were dark-adapted overnight (16 hr), anesthetized with sodium pentobarbital (50 mg/kg), and the eyes were enucleated in dim red light. Retinas were carefully dissected away from the pigment epithelium and sections of these retinas were quickly frozen over dry ice and freeze-dried for 16 hr. Some of the dissected retinas and remaining eyecups were fixed, embedded, sectioned, and examined under the microscope to ascertain that the outer segment layer was intact and that no debris remained in the eyecup. Examination of the retinas indicated that there was little if any pigment epithelial contamination. After freeze-drying, retinas were weighed,. homogenized, and sonicated in a solution of 10 mg/ml sulfosalicylic acid. The insoluble debris was pelleted by centrifugation (4,000 X g, 15 min) and taurine content in the supernatant was analyzed with a Beckman Amino Acid Analyzer (Beckman Inst; Palo Alto, CA) as previously described. 5 In some experiments, freezedried retinas were microdissected into a photoreceptor cell and an inner retina layer. The percentage of taurine within a given photoreceptor cell layer was calculated from the taurine values within corresponding photoreceptor cell and inner retina layers. The concentration of taurine was expressed either as nmol/ mg dry weight or as mmol/kg wet weight (ie, mm) based on analyses that showed that 1 mg dry weight is equivalent to 5 mg wet weight of retina. As a measure of photoreceptor numbers, the number of rows of photoreceptor cell nuclei were counted within two regions (central and peripheral) of the retina in a total of 16 normal and 31 affected setters. Dogs were anesthetized with sodium pentobarbital and the eyes enucleated at various ages (normal, 13 postnatal days to 2 yr; affected, 10 postnatal days to 1 yr). The entire eyecup or a segment of eyecup (either the temporal half of the globe including the optic disc or a retinal strip extending superior to and including the optic disc in the vertical meridian) were fixed in 2.5% glutaraldehyde in cacodylate buffer, postfixed in 2% osmium tetroxide, and embedded in an epoxy resin. One-micron-thick plastic sections, stained with azure II/methylene blue, were taken from the peripheral and/or central retinal region. Within each section, the number of rows of photoreceptor cell nuclei was counted in three locations (center and two ends) and the average of these three values was considered representative for the mean number of rows of photoreceptor nuclei in that region. Parallel studies were conducted with mice homozygous for the rd (retinal degeneration) gene. Affected mice (initially obtained from R. L. Sidman) and control mice (C57 black from Jackson Laboratories, Bar Harbor, ME) were bred in our colony. Mice of various ages were killed in dim red light after 16 hr of dark-adaptation, retinas were carefully dissected from the pigment epithelium and taurine concentration was determined as above. Progression of photoreceptor cell degeneration in rd mice was monitored by analysis of retinal DNA content as previously described. 7 The findings in Irish setters and rd mice were compared with those in RCS rats. Previous results with retinas of RCS rats 7 were replotted, in parallel, with results from Irish setters and rd mice, in terms of percentages of normal adult retinal taurine concentrations versus percentages of adult photoreceptor cell numbers, at defined stages of photoreceptor cell development and degeneration. The time course of development of photoreceptor cell function was obtained from published reports on the electroretinogram (ERG) in the normal and affected animals of the three species. 7 ' Results Counts of rows of nuclei within the outer nuclear layer indicate that normal adult numbers of photoreceptor cell nuclei were present in retinas of both normal and affected Irish setters between 10 and 26 days; normal, 11.4 ± 1.0 rows (mean ±SD for three retinas, six regions); affected 10.8 ± 1.6 rows (for nine retinas, 17 regions) (Fig. 1, top). Loss of photoreceptor nuclei progressed rapidly in affected setters between 26 and 45 days and more gradually thereafter. The numbers of photoreceptor cell nuclei were reduced to approximately 50% of normal at 50 days and to 20-30% of normal at 128 days. The loss of photoreceptor cells continued throughout the extent of the study (346 days of age) and the numbers of photoreceptor nuclei became reduced to 12-20% of normal at 192 days and 3-10% of normal at 346

3 No. 5 TAURINE IN IRISH SETTERS WITH ROD-CONE DYSPLASIA / Schmidr ond Aguirre 681 days (not shown). In retinas of normal setters a slight decline in photoreceptor cell numbers was observed between 25 days and 2 years of age. Regression analyses (58 data points for affected, 14 for normal) indicated that in the affected, the decline in numbers of photoreceptor nuclei was correlated with age (r = 0.94), whereas in the normal the slope was not significantly different from zero. The concentration of taurine within retinas of both normal and affected setters increased similarly between 10 and 26 postnatal days (Fig. 1, bottom). In normal retinas, taurine levels continued to increase and reached adult values at 45 days of age (205 ± 20 nmol/mg retina dry weight; mean ± SD). In retinas of affected setters, however, the concentration of taurine did not increase beyond the 26th day and remained at that value at 35 and 45 days of age. The concentration of taurine decreased significantly after 45 days of age and became reduced to 40-50% of normal at 140 days. It continued to decline and was reduced to 30-35% of normal by 346 days of age (not included on graph). Analyses of microdissected retinas (Table 1) showed that as in the whole retina, within the surviving photoreceptor cells of affected setters, the concentration of taurine remained constant between 26 and 45 days of age during the initial stages of photoreceptor cell degeneration. In contrast, in the normal photoreceptor cell layer, taurine levels increased to adult values of 50 mm at 45 days of age. In retinas of normal setters, the photoreceptor cell layer contained 59% of the retinal taurine at 26 days and 74% at 45 days. In retinas of affected setters, the concentration of taurine within the photoreceptor cell layer was comparable to that in the normal at 26 days but failed to increase thereafter, and by 45 days the percentage of retinal taurine within the photoreceptor cell layer was reduced to 40%. Within the inner retina, taurine concentrations (24-27 mm) remained unchanged between 26 and 45 days in both normal and affected setters. These taurine concentrations for inner retina were similar to, although slightly higher than, values obtained for six retinas analyzed from nearly photoreceptorless 2-year-old affected setters (16-22 mm). Comparison of developmental changes in retinal taurine concentration in normal animals and three species with photoreceptor cell degeneration (Irish setters with rod-cone dysplasia and rd/rd mice and RCS rats) revealed that in normal retinas, taurine levels reached adult values at a time corresponding to the development of adult ERG function (Fig. 2, arrows). In affected Irish setters and rd/rd mice, retinal taurine concentrations were comparable to AGE (doys) Fig. 1. Changes in the number of rows of photoreceptor cell nuclei in the outer nuclear layer {top) and retinal taurine concentration (bottom) as a function of age in normal Irish setters (O, control; dashed lines) and in setters with rod-cone dysplasia (A, affected; solid lines). The rows of photoreceptor cell nuclei were counted within the central and a peripheral region of the retina in most of the animals and mean values for each region have been plotted. The data for central and peripheral regions are expressed with the same symbols as these values were not significantly different from each other (each value within 10% of the mean of the 2 values for each given animal). The values for taurine concentration (nmol/mg dry weight) represent the mean ± SD (bars) for analysis of retinas from 4 to 8 different dogs at each age. normal preceding onset of photoreceptor cell degeneration but failed to increase thereafter and remained at a constant level until 40-50% of the photoreceptor Table 1. Concentration of taurine within the photoreceptor cell layer and inner retina at early stages of photoreceptor cell degeneration in Irish setters with rod-cone dysplasia Irish Setters Affected 26 days 45 days Normal 26 days 45 days Photoreceptor cell layer 34 ±6 38 ±5* 36 ± 6 50 ±9 Concentration (mm) Inner retina 23 ± 5 24 ± ±5 The concentration of taurine is expressed 10" 3 M and the values represent the mean ± SD for 4-6 analyses of microdissected retinas from affected and normal Irish setters. The concentration of taurine within these layers was calculated from the percentage of taurine within each corresponding photoreceptor cell and inner retina, the data in Figure 1, and from the volume of the photoreceptor layer. These latter values were based on analyses showing that I mg dry weight of retina was equivalent to a 5 /il volume and on data showing that the photoreceptor layer comprised about 50% of the retinal weight in 26-day-old normal and affected setters, 60% in 45-day-old normal setters and 30% in 45-day-old affected setters. * Significantly lower than corresponding normal value (P < 0.005).

4 682 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1985 Vol % PHOTORECEPTOR LOSS IN AFFECTED (%of control) Fig. 2. Retinal taurine concentration expressed as a percentage of normal adult values in three species of animals affected with hereditary retinal degeneration (affected; A) and appropriate normal control animals (O) vs photoreceptor cell numbers. The initial portions of the graphs represent early developmental periods during which all (100%) of the adult photoreceptors are present in both normal and affected animals (dog, days; mouse, 7-11 days; rat, days). The latter portions of the graphs represent periods following onset of photoreceptor cell degeneration in the three affected species. Loss of photoreceptor cells was calculated from counts of rows of photoreceptor nuclei (dog) or from analysis of retinal DNA content (mouse and rat). Photoreceptor cell death proceeded at different rates in the three affected species and 70-80% of the photoreceptor cells were lost by about 128 days in dogs, days rd mice and days in RCS rats. No significant loss of photoreceptor cells was noted in the control animals. Data points for each of the three species represent corresponding ages for normal and affected animals. The arrows designate the time at which the normal adult ERG function is attained in each of the normal species (dog, 45 days; mouse, 18 days; rat, 23 days). The values represent the mean ± SD for 4-8 analyses. cells had degenerated. Thereafter, taurine levels declined to less than 30% of control as the photoreceptor layer was reduced to 10-15% of normal cell numbers. In RCS rats, taurine levels reached adult values comparable to normal, but after onset of photoreceptor cell degeneration, taurine levels became reduced in parallel with photoreceptor cell loss. Discussion These studies show that in Irish setters with hereditary rod-cone dysplasia retinal taurine levels became reduced secondary to the loss of photoreceptor cells. During early stages of retinal development (between 10 and 26 days), normal adult numbers of photoreceptor cells were present and taurine levels increased similarly within photoreceptor cells of both normal and affected setters. In normal photoreceptor cells, taurine levels continued to increase and reached adult values in parallel with the previously reported course of development of normal adult ERG function. 15 In the affected setters, on the other hand, taurine levels failed to increase after onset of photoreceptor cell degeneration and began to decline after 45 days of age after 40-50% of the photoreceptor cells had degenerated. These results can be contrasted with the findings in cats with nutritionally induced taurine deficiency, in which a reduction in retinal taurine content was shown to precede photoreceptor cell death. 4 A comparison can be drawn between the early onset diseases in Irish setters with rod-cone dysplasia and in mice homozygous for the rd gene; in each of these animals, a defect that precedes photoreceptor cell degeneration has been shown to be a deficiency in the activity of a photoreceptor cell specific cyclic nucleotide phosphodiesterase. 8 " 1012 Although taurine levels increased during retinal development in both affected setters and rd mice, they failed to reach normal adult values (Fig. 2). In contrast, in RCS rats in which photoreceptor cell degeneration occurs after completion of outer segment development, 713 taurine levels reached adult values at days in parallel with the onset of normal adult ERG function. 7 It is interesting to note that in all these animals, even though taurine is concentrated within the photoreceptor cell layer, especially within the region of the outer nuclear layer, 13 taurine levels only reached their maxima in parallel with the completion of outer segment development and onset of adult photoreceptor function. In summary, the findings in this study exclude a role for taurine in the etiology of photoreceptor cell degeneration in Irish setters with rod-cone dysplasia. This conclusion also agrees with the observations in rd mice 6 and RCS rats, 7 and the results indicate that in all three of these animal models of hereditary retinal degenerations, reductions in retinal taurine occur secondary to the loss of photoreceptor cells. Key words: photoreceptor degeneration, taurine, Irish setters with rod-cone dysplasia, rd mice, RCS rats References 1. Orr HT, Cohen AI, and Lowry OH: The distribution of taurine in the vertebrate retina. J Neurochem 26:609, 1976.

5 No. 5 TAUPJNE IN IRISH SETTERS WITH ROD-CONE DYSPLASIA / Schmidr and Aguirre Kennedy AJ, Neal MJ, and Lolley RN: The distribution of amino acids within the rat retina. J Neurochem 29:157, Voaden MJ, Lake N, Marshall N, and Morjaria R: Studies on the distribution of taurine and other neuroactive amino acids in the retina. Exp Eye Res 25:249, Schmidt SY, Berson EL, and Hayes KC: Retinal degeneration in cats fed casein. I. Taurine deficiency. Invest Ophthalmol 15: 47, Schmidt SY, Berson EL, Watson G, and Huang C: Retinal degeneration in cats fed casein. III. Taurine deficiency and ERG amplitudes. Invest Ophthalmol Vis Sci 16:673, Orr HT, Cohen AI, and Carter JA: The levels of free taurine, glutamate, glycine and 7-amino butyric acid during the postnatal development of the normal and dystrophic retina of the mouse. Exp Eye Res 23:377, Schmidt SY and Berson EL: Taurine uptake in isolated retinas of normal rats and rats with hereditary retinal degeneration. Exp Eye Res 27:191, Aguirre G, Farber DB, Lolley RM, Fletcher RT, and Chader GJ: Rod-cone dysplasia in Irish setters: a defect in cyclic GMP metabolism in visual cells. Science 201:1133, Aguirre G, Farber DB, Lolley RN, O'Brien P, Alligood J, Fletcher RT, and Chader GJ: Retinal degenerations in the dog. III. Abnormal cyclic nucleotide metabolism in rod-cone dysplasia. Exp Eye Res 35:625, Farber DB and Lolley RN: Enzymic basis for cgmp accumulation in degenerative photoreceptor cells of mouse retina. J Cyclic Nucleotide Res 2:139, Noell WK: Studies on visual cell viability and differentiation. Ann NY Acad Sci 74:337, Schmidt SY and Lolley RN: Cyclic nucleotide phosphodiesterase: an early defect in inherited retinal degeneration of C3H mice. J Cell Biol 57:117, La Vail MM: Analysis of neurological mutants with inherited retinal degeneration. Invest Ophthalmol Vis Sci 21:638, Lolley RN and Farber DB: Cyclic nucleotide phosphodiesterases in dystrophic rat retinas: guanosine 3',5' cyclic monophosphate anomalies during photoreceptor cell degeneration. Exp Eye Res 20:585, Buyukmihci N, Aguirre G, and Marshall J: Retinal degenerations in the dog. II. Development of the retina in rod-cone dysplasia. Exp Eye Res 30:575, 1980.