Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimer's disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

Changes in protein phosphorylation induced by phagocytic challenge were identified in cultured rat retinal pigment epithelium (RPE) following exposure to isolated rat rod outer segments (ROS) or to polystyrene latex microspheres (PSL). RPE phosphoproteins were characterized based on molecular weight and isoelectric point and 32P incorporation into phosphoproteins was quantified by digitized image analysis of two-dimensional gel autoradiograms. Changes in the phosphorylation of RPE proteins were determined by comparing 32P gel data from phagocytically challenged cultures with control cultures. ROS-specific changes were defined as those occurring only in response to ROS while nonspecific changes were those associated with either ROS or PSL phagocytosis. A parallel study was conducted to identify those proteins which also show increased phosphorylation following protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate. ROS-specific increases in the phosphorylation of 2 RPE proteins were found, 1 of which also showed an increase with PKC activation. Nonspecific increases included the phosphorylation of 11 RPE proteins, 10 of which were also phosphorylated with PKC activation. ROS-specific decreases were observed in 12 RPE phosphoproteins while 3 proteins showed nonspecific decreases in their phosphorylation. These findings demonstrate that phagocytic challenge of the RPE with either specific or nonspecific particles is linked to the activation of phosphatases and kinases and that activation of PKC may play a role in phagocytosis of both particle types. The identification of two distinct groups of changes in phosphorylation supports the hypothesis that different pathways exist for phagocytosis of ROS-specific and nonspecific particles by the RPE.

Opsin phosphorylation in light was detected in three retinas from autopsy eyes with retinitis pigmentosa (RP) including one with sex-linked RP and two with autosomal recessive RP, that were studied at postmortem intervals of 1-4 hr. In these retinas from RP eyes, opsin phosphorylation in light was reduced compared with that in normal human retinas, a finding that is compatible with reduced amounts of opsin due to extensive loss of photoreceptor cells. ATP and GTP levels, although reduced below normal, also were easily detectable, supporting the idea that the reduction in opsin phosphorylation was due to loss of photoreceptor cells and not to a reduced capacity for energy metabolism. These findings in these RP retinas contrast with those in rd mice and Irish setters with rod-cone dysplasia in which a failure of opsin phosphorylation has been detected prior to onset of photoreceptor cell degeneration.

Interphotoreceptor matrix (IPM) proteins from a wide range of vertebrate species were examined by gel electrophoresis. Extensive similarities in the banding patterns of the proteins were found. S antigen, serum albumin and interphotoreceptor retinoid-binding protein (IRBP) were identified immunochemically. The latter two proteins dominate the IPM obtained from the apical surface of the retinal pigment epithelium, whereas IPM prepared from the retina washes contains IRBP plus outer-segment components including S antigen. IRBP is present in IPM from the all-cone lizard (Anolis) eye, as well as from rod- and cone-dominant animals. The ontogeny of IRBP in chick IPM is different from that of serum albumin in age of onset and rapidity of development. Comparison between Royal College of Surgeons rat IPM and normal rat IPM showed that several proteins are changed in amount. This study is a step toward a functional characterization of components common to the IPM of all vertebrates.

Incubation of confluent cultures of rat retinal pigment epithelium (RPE) with 32P-orthophosphate resulted in the incorporation of 32P into proteins, RNA and the nucleoside phosphates ADP, GDP, ATP and GTP. RPE cultures incubated with phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, did not significantly change the incorporation of 32P into total protein, RNA or the nucleoside phosphates ADP, GDP, ATP and GTP. However, PMA exposure specifically increased phosphorylation of five proteins with molecular weights of 80 kilodaltons (K), 56K, 35K, 33K, and 29K having isoelectric points between 4.3 and 6.5. PMA treated cultures also showed dephosphorylation of two proteins having molecular weights of about 33K. The observed increase in 80K phosphorylation suggests that protein kinase C is present and activated by PMA in the RPE.

Opsin, the alpha-subunit of transducin, S-antigen, interphotoreceptor retinoid-binding protein (IRBP) and cathepsin D were assessed in autopsy eyes from patients with retinitis pigmentosa (RP) and normal autopsy eyes. Immunochemical methods were used to determine the presence of these proteins on Western blots of retinal homogenates from five RP donors and on blots of interphotoreceptor matrix (IPM) preparations from six other RP eyes. The amounts of immunoreactive opsin, S-antigen, alpha-transducin, and IRBP appeared below normal in retinas from RP eyes. All six IPM samples from patients with advanced RP had reduced amounts of S-antigen and no detectable IRBP or transducin. Cathepsin D (an RPE protein) was present in IPM or RP eyes in amounts comparable to that in IPMs from normal eyes. Small amounts of cathepsin D were also detected in retinas from both normal and RP eyes. These studies show that proteins specific to the photoreceptor-pigment epithelium complex in normal eyes can be detected in autopsy eyes from patients with RP and suggest that the observed reductions in photoreceptor-specific proteins occur as a consequence of photoreceptor loss.

Kinetically distinct classes of cyclic AMP (cAMP) and cyclic GMP (cGMP) phosphodiesterase activities (PDEs) were detected in homogenates of cultured pigment epithelium (PE) from both normal and Royal College of Surgeons (RCS) rats. PDE activities with apparent low Michaelis constants (Low Km cAMP- and cGMP-PDEs) were associated with the supernatant, while PDE activities with apparent high Michaelis constants (high Km cAMP- and cGMP-PDEs) were slightly higher in the pellet than in the supernatant after ultracentrifugation (100,000 g). Activity of the low Km PDEs was significantly reduced while that of high Km PDEs was not affected by known inhibitors of PDE. In both normal and RCS rat PE low Km PDEs required calcium and magnesium ions for optimal activity while the high Km PDEs required neither. In homogenates of cultured RCS rat pigment epithelium (PE), the kinetic parameters for cAMP- and cGMP-PDEs were comparable to normal, with the exception of the Km value of the low Km cGMP-PDE. This Km value was two-fold higher in the RCS compared with the normal (indicative of a reduced affinity for cGMP). It remains to be determined if the reduced affinity for cGMP in the RCS PE is related to the genetic defect which is expressed as a deficiency in the phagocytosis of outer segments by these cells.

Melanin concentrations were analyzed in the pigment epithelium of 61 postmortem normal human eyes from donors 14-97 yr of age. The content of soluble melanin in the pigment epithelium declined with age from the highest values of 95 micrograms/mg in the 14-50 yr age group to the lowest values of 22 micrograms/mg dry weight in the over 70 yr age group. In 16 of these eyes, regional measurements revealed that melanin concentrations were lowest in the macula-perimacular area (a region 5-8 mm in diameter centered on the fovea), were higher in the mid-periphery, and were highest in the far periphery. In the macular region, melanin concentrations were similarly low at all ages, whereas, in the mid- and far-peripheral regions, melanin concentrations decreased with age. These studies demonstrate age-related reductions and regional variations in melanin concentrations in the pigment epithelium of postmortem normal human eyes, and establish a baseline for future comparison with donor eyes from patients with hereditary retinal degenerations.

A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.

Annals of the New York Academy of SciencesVolume 463, Issue 1 p. 224-226 Light Stimulates Messenger RNA Synthesis in Photoreceptor Cells of the Rat SUSAN Y. SCHMIDT, SUSAN Y. SCHMIDT Berman-Gund Laboratory for the Study of Retinal Degenerations Department of Ophthalmology Harvard Medical School Massachusetts Eye and Ear Infirmary Boston, Massachusetts 02114Search for more papers by this authorJANET C. BLANKS, Corresponding Author JANET C. BLANKSb Department of Ophthalmology and Anatomy and Cell Biology University of Southern California School of Medicine and Estelle Doheny Eye Foundation Los Angeles. California 90033Search for more papers by this authorMICHAEL A. SANDBERG, MICHAEL A. SANDBERG Berman-Gund Laboratory for the Study of Retinal Degenerations Department of Ophthalmology Harvard Medical School Massachusetts Eye and Ear Infirmary Boston, Massachusetts 02114Search for more papers by this author SUSAN Y. SCHMIDT, SUSAN Y. SCHMIDT Berman-Gund Laboratory for the Study of Retinal Degenerations Department of Ophthalmology Harvard Medical School Massachusetts Eye and Ear Infirmary Boston, Massachusetts 02114Search for more papers by this authorJANET C. BLANKS, Corresponding Author JANET C. BLANKSb Department of Ophthalmology and Anatomy and Cell Biology University of Southern California School of Medicine and Estelle Doheny Eye Foundation Los Angeles. California 90033Search for more papers by this authorMICHAEL A. SANDBERG, MICHAEL A. SANDBERG Berman-Gund Laboratory for the Study of Retinal Degenerations Department of Ophthalmology Harvard Medical School Massachusetts Eye and Ear Infirmary Boston, Massachusetts 02114Search for more papers by this author First published: May 1986 https://doi.org/10.1111/j.1749-6632.1986.tb21553.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 SCHMIDT, S. Y. 1983. J. Cell Biol. 97: 824– 831. 2 HOLLYFIELD, J. G. & S. F. BASINGER. 1980. Neurochemistry 1: 103– 112. 3 SCHMIDT, S. Y., J. C. BLANKS & M. A. SANDBERG. 1985. Exp. Eye Res. 41: 159– 170. 4 TAYLOR, J. M. 1979. Ann. Rev. Biochem. 48: 681– 717. 5 HILL, R. E., K. LEE & F. T. KENNEY. 1981. J. Biol. Chem. 256: 1510– 1513. 6 POTTER, E., A. K. NICOLAISEN, E. S. ONG, R. M. EVANS & M. G. ROSENFELD. 1981. Proc. Natl. Acad. Sci. 78(11): 6662– 6666. 7 CUPELLO, A. & H. HYDEN. 1976. Brain Res. 114: 453– 460. 8 OLNEY, J. W. 1982. Retina 2: 341– 359. 9 POTTS, A. M., R. W. MODRELL & C. KINGSBURY. 1960. Am. J. Ophthalmol, 50: 900– 907. 10 POTTS, A. M. 1965. In Biochemistry of the Reinta. C. N. GRAYMORE, Ed. Academic Press. New York . pp. 155– 162. 11 COHEN, A. I. 1967. Am J. Anat. 120: 319– 356. 12 LAVAIL, M. M. 1981. Invest. Ophthalmol. Vis. Sci. 21: 638– 657. Volume463, Issue1Second Colloquium in Biological Sciences: Intercellular Communication and Cell Surface ReceptorsMay 1986Pages 224-226 ReferencesRelatedInformation

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.

Incorporation of [3H]-cytidine into messenger RNAs (polyadenylated RNAs) was enhanced in light in isolated intact photoreceptor cells of the rat. The increase in polyadenylated (PolyA+)RNAs appeared selective relative to other photoreceptor RNAs since incorporation of [3H]-cytidine into this fraction was up to 10-fold higher while labeling of total cellular RNAs was only two- to three-fold higher in light compared with dark. The photoreceptor cells were isolated in vivo through destruction of inner retina neurons with injections of combinations of neurotoxic substances during early postnatal development. The photoreceptor cells attained normal adult morphology and function: the alpha-wave of the electroretinogram, as well as the thickness of the outer nuclear layer and the length of photoreceptor inner and outer segments, were found to be within the normal range at 4 and 10 weeks of age. In addition to RNA synthesis, such photoreceptor cell preparations when incubated in vitro demonstrated a capacity for regulating light-dependent sodium fluxes comparable to that within the intact retina. The potential usefulness of this model for exploring the molecular biology of photoreceptor cells is discussed.

Incorporation of labeled precursors (glycerol, glucose, or 32Pi) into phosphatidylinositol (PI) was 2-3-fold higher in rat retinas incubated in light compared to those in dark. During brief (30 min) incubations with labeled glycerol, there was a selective increase in the radioactivity associated with PI and phosphatidic acid, whereas, upon longer (60 min) incubations, synthesis of other lipids was also enhanced in light. Accumulation of the precursors was similar in light or dark throughout 60 min of incubation. Phosphorylation of PI to triphosphoinositide (TPI) was also enhanced in light, and, in both light and dark, up to 40-50% of the total 32Pi incorporated was associated with TPI. A model is proposed for PI metabolism based on two pathways: 1) a PI cycle representing synthesis and turnover of PI, and 2) phosphorylation of PI to TPI and possible hydrolysis of TPI. Light stimulation was shown to enhance the synthesis of PI within the photoreceptor cell layer and truncated rods and to increase the phosphorylation of PI to TPI within the inner retina and synaptosomes. Parallel studies with retinas from Royal College of Surgeons rats with advanced photoreceptor cell degeneration and intact inner retina showed that loss of the photoreceptor cells did not affect the capacity for phosphorylation of PI to TPI, but was associated with reduced capacity for PI synthesis. These results provide evidence that light stimulation affects different pathways of PI metabolism within different cells of the retina.

During brief (30-min) incubations, isolated rat retinas accumulated [3H]cytidine, converted it to cytidine triphosphate (CTP), and incorporated it into RNA and cytidine diphosphate-diacylglyceride (CDG), a phospholipid precursor of phosphatidylinositol (Pl). Labeled CTP, RNA, and CDG contents were found to be two- to three-fold higher in photoreceptor cells than in cells of the inner retina. Autoradiograms showed that, within photoreceptor cells, silver grains representing RNA were concentrated over the nuclei in dark and light, while silver grains representing CDG were concentrated over the inner segments only after incubation in dark. The formation of labeled CTP and the synthesis of RNA were enhanced in light, while labeled CDG levels became reduced in light concurrent with an increase in the incorporation of labeled inositol into Pl. The 3H-labeled CDG content, however, was increased two- to fourfold in light in the presence of actinomycin D, and autoradiograms show a heavy concentration of silver grains over the inner segments of photoreceptor cells. These findings establish a role for cytidine nucleotides in photoreceptor cell metabolism and in light-dependent increases in RNA and Pl synthesis. Furthermore, the observations indicate that a competition may exist in light for cytidine or CTP and suggest that availability of cytidine for CDG synthesis may have a regulatory role in Pl metabolism within the photoreceptor cells.

Retinas of postmortem human donor eyes retained the high-affinity mechanism for uptake of 3H-taurine, the capacity to synthesize rhodopsin from 14C-amino acids, and the ability to incorporate inorganic 32P-phosphate into rhodopsin with exposure to light for 4 to 4 1/2 hr. These processes declined at a rate of about 16% to 19% per hour between 2 and 4 1/2 hr after death. Parallel studies with rats maintained after death at room temperature (22 degrees C) showed that all three processes declined linearly at rates of 8% to 12% per hour. In rats maintained after death at body temperature (37 degrees C) or on ice (4 degrees C), the rates of decline in rhodopsin synthesis were respectively 20% to 22% and 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from la 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from la 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from labeled precursors. These findings in human and rat eyes show that photoreceptor cells can perform energy-requiring processes for several hours after death.

Patients with gyrate atrophy of the choroid and retina have myopia, constricted visual fields, elevated dark adaptation thresholds, small or nondetectable ERGs, and chorioretinal atrophy. Biochemical abnormalities include hyperornithinemia, hypolysinemia, hyperornithinuria, an unknown amino compound in the urine, and virtual absence of OKT activity in extracts of cultured skin fibroblasts. Extracts of cultured skin fibroblasts from one patient studied in our laboratory showed an increase in OKT activity with increasing concentrations of vitamin B6 in the assay medium; this patient also showed some biochemical responsiveness within three weeks to 300 mg/day or orally administered vitamin B6. Three patients whose fibroblasts did not show increased OKT activity in vitro with increasing vitamin B6 did not respond in vivo to 300 mg/day of vitamin B6 over the same period. All four patients continue to be evaluated with larger doses of this vitamin. It remains to be established if long-term treatment with vitamin B6 will stabilize the course of the chorioretinal degeneration for at least some patients with this disease.