Susan Y. Schmidt

Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimers 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.

Taurine uptake in isolated retinas of normal rats and rats with hereditary retinal degeneration

Abstract Kinetic analysis reveals two processes for the uptake of taurine in the isolated rat retina. One process, designated as a high affinity uptake mechanism, has an apparent Michaelis constant ( K m ) of 50 μ m and a maximum velocity of uptake ( V max ) of 0·3 nmol/mg dry wt/min. The other process, designated as a nonsaturable uptake mechanism, has a rate constant of 0·53 nmol/mg dry wt/min/m m taurine in the medium. Retinas from normal rats, studied at ages 22–180 days, and from Royal College of Surgeons (RCS) rats with normal or reduced numbers of photoreceptor cells, studied at ages 22–45 postnatal days, show both mechanisms at a time when photoreceptor cell function is detectable in vivo with the electroretinogram (ERG). Retinas from 180-day-old photoreceptorless RCS rats retain the nonsaturable uptake mechanism for taurine but do not show the high affinity uptake mechanism at a time when the number of photoreceptor cells is greatly reduced and no photoreceptor function can be detected with the ERG. The high affinity uptake mechanism is selectively lost in isolated normal rat retinas with mechanical disruption of photoreceptor cells and is inhibited to a greater extent than the nonsaturable mechanism by ouabain and reduced temperature. These studies support the idea that the high affinity uptake mechanism for taurine in rat retinas depends on the presence of viable photoreceptor cells.

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light‐dependent increase in [3H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [3H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light‐enhanced incorporation of [3H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [3H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus‐enhanced turnover of PI in some tissues, this light‐enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (22Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of 22Na in light but not in dark.

Taurine fluxes in isolated cat and rat retinas: Effects of illumination

Abstract Taurine is taken up by isolated cat retinas incubated in an oxygenated medium in the light or in the dark. Onset and cessation of illumination are associated with a prompt transient release followed by reuptake of taurine; in contrast, glycine is gradually released only with onset of illumination. The uptake of taurine and the light evoked release of taurine followed by reuptake are inhibited by reduction in temperature, iodoacetate, ouabain, and absence of glucose. Similar light-evoked taurine fluxes are observed in the isolated retinas from normal rats and 30-day-old RCS rats but cannot be demonstrated in the photoreceptorless retinas from 180-day-old RCS rats. These findings support the idea that the effects of illumination on taurine fluxes depend on the viability of photoreceptor cells.

Ocular and biochemical abnormalities in gyrate atrophy of the choroid and retina.

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.

Effects of IBMX on the ERG of the isolated perfused cat eye

Full-field electroretinograms (ERGs) were recorded from isolated cat eyes perfused through the ophthalmociliary artery with isobutylmethylxanthine (IBMX), an inhibitor of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase. Low doses of IBMX (0.1-0.3 mM) produced decreased rod ERG amplitudes at low stimulus luminances and increased rod ERG amplitudes at high stimulus luminances. A high dose of IBMX (1.0 mM) initially produced the same effect as the low doses and then led to decreased rod ERG amplitudes at all stimulus luminances. Perfusion with IBMX also resulted in elevations in the semi-saturation luminance (sigma), delayed rod a-wave latencies, delayed rod a-wave and b-wave implicit times, and reduced rod a-wave slopes. Eyes perfused with IBMX (1.0 mM) were also found to have elevated levels of retinal cyclic GMP. These effects of IBMX on the rod ERG are considered in the context of previously described ERGs in selected cases of human retinal degeneration.

Phagocytic challenge induces changes in phosphorylation of retinal pigment epithelium proteins

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.

Comparison of Proteins in the Interphotoreceptor Matrix of Vertebrates

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.

High-affinity uptake of [3H]taurine in isolated cat retinas: Effects of Na+ and K+

Isolated cat retinas have a high-affinity mechanism for uptake of [3H]taurine associated with the photoreceptor cells and a non-saturable mechanism for uptake of [3H]taurine associated with the inner retina. Retinas from slightly taurine-deficient cats show increased affinity for [3H]taurine and increased maximum velocity of uptake while the nonsaturable mechanism for taurine uptake is not affected by taurine deficiency. The Vmax of the high-affinity uptake mechanism is enhanced when Na+ concentrations are increased or when K+ concentrations are reduced in the incubation medium. In contrast with the above findings neither Na+ nor K+ affects uptake of taurine by the nonsaturable mechanism.

Cyclic nucleotide phosphodiesterases in cultured normal and RCS rat pigment epithelium: Kinetics of cyclic AMP and cyclic GMP hydrolysis **

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.