In Vitro Phosphorylation in Isolated Horizontal Cells of the Fish Retina: Effects of the State of Light Adaptation

Horizontal cells, which are second-order neurons of the vertebrate retina, exhibit synaptic plasticity governed by light and dark adaptation. We have investigated the alterations in the protein phosphorylation patterns of isolated carp ( Cyprinus carpio ) horizontal cells in relation to their state of light adaptation by using an in vitro phosphorylation assay and compared the resulting data with protein synthesis patterns of the whole retina. Phosphoproteins and [³⁵S]methionine-labelled proteins were analysed by one- and two-dimensional gel electrophoresis followed by autoradiography. The state of light adaptation significantly affected the in vitro phosphorylation of horizontal cell proteins with molecular weights of 68, 56/58, 47, 28 and 15 kDa, but had no effect on the protein synthesis of retinal proteins. In the light the most prominent increase of ³²P incorporation was observed in the 47 kDa protein. The biochemical properties of this protein closely resembled those of the growth-associated GAP-48, found in the fish retina. In addition, the phosphorylation of horizontal cell homogenates in the presence of protein kinase activators such as cyclic AMP, calcium, calmodulin and phospholipids revealed that horizontal cells of the fish retina contain cyclic AMP-, calcium/calmodulin- and calcium/phospholipid-dependent protein kinase activity resulting in the phosphorylation of several horizontal cell proteins, including the phosphoproteins which were affected by the state of light adaptation.     

Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery.

Adult rhesus monkeys were subjected to complete cerebral ischemia for one hour and subsequent recirculation for up to 24 h. Animals with signs of functional recovery (e.g. spontaneous EEG activity) exhibited a partial replenishment of cellular energy sources (ATP, phosphocreatine) and a progressive normalization of cerebral lactate levels. Glucose and pyruvate concentrations showed a transient increase over control values during the early stages of postischemic recirculation. Monkeys without functional recovery lacked a significant resynthesis of energy-rich compounds; adenine nucleotides continued to decrease and lactate concentrations were higher than in animals subjected to ischemia without recirculation. Cerebral polysome profiles remained unaltered during the ischemic period but in all animals a marked disaggregation of polyribosomes with a concomitant increase in ribosomal subunits occurred after the onset of recirculation. In monkeys with indications of functional recovery these changes were reversible but a normal polysome profile was only observed after 24 h of recirculation. The results obtained indicate a postischemic depression of protein synthesis due to an inhibition of peptide chain initiation. After recirculation of the brain for 3-6 h there was evidence for an induction of enzymes involved in polyamine synthesis (ornithine decarboxylase and S-adenosylmethionine decarboxylase). No changes in the activity of these enzymes were observed at the end of the ischemic period, indicating that during complete cerebral ischemia not only the synthesis but also the catabolism of proteins is inhibited.     

Endogenous neuroprotection in the retina

Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows severe ischemia. The retina is protected against the damage following severe ischemia for up to 72h after the application of IPC. However, there is no early preconditioning, i.e. protective effects starting within hours of preconditioning. The IPC stimulus consists of a brief, non-damaging period of ischemia. It results in complete preservation of retinal structure and function following ischemia, and is thus the most robust neuroprotection demonstrated in the retina to date. Release of adenosine, de novo protein synthesis, and mediators such as protein kinase C and K(+) ATP channels are required for IPC protection. Both the adenosine A1 and A2a receptors are involved. However, the molecular mechanisms for neuroprotection have not been completely described. It appears that both increased expression of protective proteins and decreased expression of pro-apoptotic proteins are involved. In addition, IPC prevents hypoperfusion following severe ischemia. Further study of the IPC phenomenon could lead to an enhanced understanding of the mechanisms of ischemic damage and its prevention in the retina.     

Distribution of specific intermediate-filament proteins in the goldfish retina

The intermediate-filament proteins expressed in the goldfish retina were investigated by immunohistochemistry and by immunoblotting. Polyclonal antibodies that previously had been raised against the goldfish optic nerve neurofilament (ON1 and ON2) and glial filament (ON3 and ON4) proteins were used in this study. Anti-ON1/ON2 antiserum reacted on a retinal immunoblot with two proteins having molecular weights and isoelectric points corresponding to those of ON1 and ON2. Histologically, the most pronounced anti-ON1/ON2 reactivity was observed in the ganglion cell layer of the goldfish retina. The anti-ON3/ON4 antiserum reacted with a single protein on a retinal immunoblot. This protein had a molecular weight and isoelectric point which corresponded to the goldfish optic nerve glial filament proteins. This anti-serum labeled horizontal cells in retina sections. Three previously unidentified goldfish visual-pathway intermediate-filament proteins sharing a molecular weight of 60K were observed on two-dimensional gels of retinal cytoskeletal proteins and on retinal immunoblots which were probed with a monoclonal antibody which recognizes an epitope common to all intermediate filament proteins. The possible existence of homologs of mammalian GFAP and vimentin in the goldfish retina was also explored. Antibodies directed against mammalian GFAP and vimentin labeled the Müller fibers and the cone horizontal cells, respectively. However, immunoblot analysis and a comparison of the two-dimensional gel electrophoresis patterns of goldfish retinal and rat spinal cord cytoskeletal proteins demonstrated a lack of goldfish proteins identical to the mammalian intermediate-filament proteins.     

Expression of basal lamina protein mRNAs in the early embryonic chick eye

Laminin, collagen IV, collagen XVIII, agrin, and nidogen are major protein constituents of the chick retinal basal lamina. To determine their sites of synthesis during de novo basal lamina assembly in vivo, we localized their mRNA expression in the eye during maximum expansion of the retina between embryonic day (E) 2.5 and E6. Our in situ hybridization studies showed that the expression pattern of every basal lamina protein mRNA in the developing eye is unique. Collagen IV and perlecan originate predominantly from the lens epithelium, whereas collagen XVIII, nidogen, and the laminin gamma 1 and beta1 chains are synthesized mainly by the ciliary body. Agrin, collagen XVIII, collagen IV, and laminin gamma 1 also originate from cells of the optic disc. The only basal lamina protein that is synthesized by the neural retina throughout development is agrin with ganglion cells as its main source. Some of the mRNAs have short, transient expressions in the retina, most notably that of collagen IV and laminin gamma 1, both of which appear in the ventral retina between E4 and E5. That most retinal basal lamina proteins originate from extraretinal tissues infers that the basal lamina proteins have to be shed from the lens, optic disc, and ciliary body into the vitreous body. The assembly of the retinal basal lamina then occurs by the binding of these proteins by cellular receptor proteins on the vitreal endfeet of the retinal neuroepithelial cells.     

Selective effects of LSD and hyperthemia on the synthesis of synaptic proteins and glycoproteins

Protein synthesis in rabbit brain was inhibited following the intravenous injection of LSD. The incorporation of [³⁵S]methionine into brain microsomal and synaptic fractions was decreased by 35–45% relative to control values. A selective increase was observed, however, in the relative labeling of a protein of molecular weight 75,000. Our previous studies have shown that LSD induces an increase in body temperature (i.e. hyperthermia) in rabbits. When LSD-induced hyperthermia was blocked the general reduction in labeling of microsomal and synaptic proteins was still apparent but the selective increase in relative labeling of the 75,000 dalton protein was not. Induction of hyperthermia by means other than LSD (i.e. elevation of ambient temperature) produced selective increases in the relative labeling of microsomal and synaptic proteins of molecular weight 75,000 and 95,000. These proteins are similar in molecular weight to two of the major ‘heat shock’ proteins whose synthesis is induced in several cultured cell lines following elevation of ambient temperature. Fractionation of [³⁵S]methionine-labeled synaptic membranes by lectin affinity chromatography and analysis of [³⁵H]fucose labeling patterns indicated that, in contrast to the general reduction in labeling of brain proteins, the synthesis of synaptic glycoproteins was not altered by LSD. The synthesis of glycosylated proteins present in other subcellular fractions was, however, reduced. These results suggest that LSD induced selective changes in the synthesis of brain proteins and that the synthesis of synaptic glycoproteins may be relatively resistant to drug administration.     

Reduced protein synthesis in diabetic retina and secondary reduction of slow axonal transport

Protein synthesis in the retina in diabetic rabbits decreased as the severity of the diabetes increased, but protein degradation and replacement were not affected. The reduction in the amount of orthograde slow axonal transport in diabetic rabbits closely paralleled the reduction in the amount of fast axonal transport and protein synthesis in the retina. The reduction in slow flow is most probably a simple reflection of reduced protein synthesis in the retinal ganglion cells. The relationships among reduction in axon diameter, volume of perikaryon, slow axonal transport and diabetic neuropathy are discussed.     

Levels of transient gap junctions between the retinal pigment epithelium and the neuroblastic retina are influenced by catecholamines and correlate with patterns of cell production

Retinal mitosis takes place at the interface between the retinal pigment epithelium (RPE) and the neural retina. Multiple studies have highlighted the essential role that gap junction-mediated communication plays in the regulation of retinal organogenesis. Here, the localization pattern and function of the gap junction protein connexin 43 were examined in vivo in the rat at the interface between the retina and RPE during the main phases of retinal cell production. Connexin 43 was expressed at this site from E15 onward, and levels were subsequently temporally regulated. When Cx43 protein levels were reduced experimentally, by using antisense oligodeoxynucleotides, mitotic activity in the retina decreased significantly. Conversely, in the hypopigmented eye elevated mitotic levels were associated with a significant increase of connexin 43. Both excess protein levels and elevated mitosis were corrected by the in vivo administration of L-DOPA (a dopamine precursor and intermediary compound in the melanin synthesis pathway). These findings suggest that connexin 43-mediated communication between the retina and RPE is essential for the correct pacing of retinal organogenesis. Furthermore, this pathway may be gated by levels of ocular catecholamines.     

Glial transcytosis of a photoreceptor-secreted signaling protein, retinoschisin

In vitro studies have clearly shown that signaling/guidance proteins can diffuse to their targets. However, it is unclear whether they can travel by diffusion in vivo, or if they are distributed in the tissue by an active mechanism. Retinoschisin, a signaling molecule related to neuropilins, is synthesized and secreted by photoreceptor cells in the outer retina; then it interacts with inner retinal cells contributing to synaptic organization and optic nerve fiber integrity. We developed an assay to examine how retinoschisin, which is secreted a distance away, reaches its inner retinal targets. We found that retinoschisin is preferentially taken up and carried into the inner retina from the retinal outer border (the photoreceptor side) by Müller cells (the main glial cells of the vertebrate retina). This transcytosis is disrupted by DL-alpha-aminoadipic acid, a Müller cell/glia-specific toxin. Our results suggest that glial uptake/transcytosis can provide an effective and precise alternative for distributing signaling molecules in the nervous system.     

Retinal astrocyte differentiation mediated by leukemia inhibitory factor in cooperation with bone morphogenetic protein 2

Retinal astrocytes and their precursor cells migrate from the optic nerve. Interleukin 6 family cytokines, whose signal transduction requires gp130, promote astrocyte differentiation in the optic nerve, though the mechanism of astrocyte differentiation in the retina has not been clarified. We found that GFAP-positive astrocytes were significantly decreased in number but that a considerable number of astrocytes were still present in gp130-deficient mouse retina. These findings suggest that gp130-dependent signaling pathways play essential roles in retinal astrocyte differentiation and that retinal astrocyte differentiation can also be promoted by other signaling pathways. We found that leukemia inhibitory factor, bone morphogenetic proteins, and their receptors are expressed in P0 retina. In addition, leukemia inhibitory factor and bone morphogenetic protein 2 synergistically promote astrocyte differentiation of retinal precursor cells isolated from P0 mouse retina. These observations demonstrated that not only gp130-dependent signaling but also bone morphogenetic proteins play essential roles in retinal astrocyte differentiation.     

Induction of heat shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post-hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley-Liss, Inc.     

Distribution of Purkinje cell-specific Zebrin-II/aldolase C immunoreactivity in the mouse,rat, rabbit,and human retina

The developmental, genetic, and biochemical similarities that have been observed between the cerebellum and retina form the basis for ongoing investigations into retinal expression of cerebellar-specific proteins. We have examined the mouse, rat, rabbit, and human retina for expression of a protein that is present in parasagittal Purkinje cell strips and that is recognized by the antibody Zebrin-II. This protein has recently been identified as a member of the aldolase C isoenzymes. Western blotting and immunocytochemistry have been used. The monoclonal antibody Zebrin-II recognized a prominent 36 kDa protein band on immunoblots of both the cerebellum and the retina of the examined species. Immunocytochemistry showed that, in the three nonhuman species, cells were stained in the ganglion cell layer (GCL). In addition, in the mouse and rabbit, cells in the inner nuclear layer (INL) were also labeled. Except for the visual streak, there were more immunopositive cells in the rabbit GCL and INL than in corresponding areas of the mouse retina. In the human, in contrast to the other species, the photoreceptor cell layer was strongly aldolase C immunoreactive. In the h all species except for the rat, the photoreceptor inner segments also displayed a weak labeling. The results show that this aldolase C isoenzyme is another protein that is selectively expressed by the cerebellum: and retina. Furthermore, the retinal expression is species specific, and this pattern seems to show a good correlation with the oxygenation level of the individual compartments. The indication that this aldolase C isoenzyme has specific developmental functions in the retina provides additional clues for our understanding of cerebellar organization. © 1994 Wiley-Liss, Inc.     

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.     

Immunopurification of an S-antigen-like protein from human platelets.

S-antigen (also named arrestin or 48K protein) is a protein abundant in photoreceptor cells of vertebrates and invertebrates. The presently known function of this protein in retina is to arrest the enzymatic cascade of phototransduction in retinal rods, through its binding to photoactivated and phosphorylated rhodopsin. Proteins closely related to S-antigen were recently demonstrated in several non photosensitive cells. In this work, we demonstrated the presence of a protein similar to retinal S-antigen with regards to its immunoreactivity with a panel of monoclonal antibodies and its molecular weight in soluble extracts of human platelets. This protein was purified by immunoaffinity chromatography using a rabbit antibody to retinal S-antigen. This S-antigen-like protein could have a regulatory function in G-protein-mediated transduction of chemical signals in platelets, similar to arrestin function in phototransduction.     

Hyperthermic pre-conditioning protects retinal neurons from N-methyl-D-aspartate (NMDA)-induced apoptosis in rat

Glutamate-induced excitotoxicity is associated with a selective loss of retinal neurons after retinal ischemia and possibly in glaucoma. Since heat shock protein (HSP) 70 is known to play a protective role against ischemic neuronal injury, which is also linked to excitotoxicity, we studied the expression of inducible (HSP72) and constitutive (HSC70) forms of HSP70 in apoptosis of retinal ganglion cells (RGCs) after intravitreal injection of 8 nmoles N-methyl-D-aspartate (NMDA), a glutamate receptor agonist. Approximately 18 h after NMDA injection, there were increased numbers of TUNEL-positive cells and cells with elevated HSP72 immunoreactivity in the retinal ganglion cell layer (RGCL), but there were no noticeable changes in HSC70 immunoreactivity. These HSPs positive cells were also Thy-1 positive, a marker for RGCs. Hyperthermic pre-conditioning, which is known to induce HSPs, given 6 or 12 h prior to NMDA injection ameliorated neuronal loss in the RGCL as counted 7 days after NMDA injection but pre-conditioning at 18 h prior to NMDA injection did not have any ameliorative effect. Quercetin, an inhibitor of HSP synthesis, abolished the ameliorative effect of hyperthermic pre-conditioning. Pre-conditioning elevated HSP72 but not HSC70 immunoreactivity and reduced the number of TUNEL-positive cells in the RGCL at 18 h. Our results suggest that intravitreal injection of NMDA induces an up-regulation of HSP72 in a time-dependent manner but not HSC70 in RGCs, indicating a stress response of HSP72 in RGCs and other inner retinal neurons after exposure to NMDA. Hyperthermic pre-conditioning given within a therapeutic window is neuroprotective to the retina against NMDA-induced excitotoxicity, likely by inhibiting apoptosis through the modulation of HSP72 expression.     

Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina

Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague–Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for β-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.     

Astrocytes in the guinea pig, horse, and monkey retina: their occurrence coincides with the presence of blood vessels

In the present study the distribution of astrocytes in the nerve fiber layer (NFL) has been studied in the sparsely vascularized retinae of the guinea pig and horse and in the richly vascularized retina of the Old World monkey (Cercopithecus aethiops) using immunocytochemical methods. In the guinea pig retina glial fibrillary acidic protein (GFAP)-positive astrocytes could not be detected. They were found, however, in the myelinated region of the optic nerve. The optic nerve head and a small retinal region immediately adjacent to it contained few vimentin-positive astrocytes. Histological sections confirmed the restriction of astrocytes to a small retinal region and showed that this is also the only retinal area that is vascularized. Astrocytes showing GFAP and vimentin immunoreactivity were absent from most of the horse retina. They were found only in a narrow zone close to the optic disc, which is also the only region of the horse retina that is vascularized. Thus, as in the rabbit retina (Schnitzer: J. Comp. Neurol. 240:128-142, 1985), in the guinea pig and horse retina astrocytes are not present ubiquitously in the NFL but coexist with blood vessels. In the monkey retina, GFAP-positive astrocytes were found ubiquitously in the NFL. Astrocytes were absent from the avascular foveal region only. It is suggested that the concurrence of retinal astrocytes and intraretinal vascularization may be a feature common to many, if not all, mammalian species.     

Regulation of indoleamine N-Acetyltransferase activity in the retina: Effects of light and dark, protein synthesis inhibitors and cyclic nucleotide analogs

The regulation of indoleamine N-acetyltransferase (NAT) in the posterior eye was investigated in vivo, and in vitro in cultured eye cups. Surgical separation of neural retinal from the retinal pigment epithelium-choroid complex indicated that NAT was localized to neural retina. The activity of retinal NAT fluctuated in vivo in a rhythmic fashion, with peak activity in the dark phase of the light-dark cycle. The rhythm of NAT activity persisted for up to 3 days in constant darkness, with a rhythmic period of approximately 25 h. The rhythm was suppressed by constant light, and could be phase-shifted by exposure to a new light-dark cycle. These observations indicate that retinal NAT activity occurs as a circadian rhythm that is entrained by light and dark. Retinas also responded to light and dark in vitro with changes of NAT activity. A significant increase in retinal NAT activity occurred in eye cups cultured in darkness during the dark phase of the light-dark cycle. This increase was completely suppressed in eye cups cultured at the same time of day in light. The dark-induced increase in NAT was completely blocked by protein synthesis inhibitors, and mimicked in light by cyclic AMP analogs.The similarity of the regulation of NAT activity in retina to that in pineal, and the possible relationship of the retinal NAT rhythm to cyclic metabolism in photoreceptors are discussed.