Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324–348) and frog (amino acids 330–354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.

Regulation of eye development by frizzled signaling in Xenopus

Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation and whether transmembrane signaling events are involved. Here we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation and can perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to that of endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx, and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Rx, and Otx2 expression and suppression of endogenous eye development. This effect can be rescued by coexpression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary, we show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Sleep modifies retinal ganglion cell responses in the normal rat

Recordings were obtained from the visual system of rats as they cycled normally between waking (W), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Responses to flashes delivered by a light-emitting diode attached permanently to the skull were recorded through electrodes implanted on the cornea, in the chiasm, and on the cortex. The chiasm response reveals the temporal order in which the activated ganglion cell population exits the eyeball; as reported, this triphasic event is invariably short in latency (5–10 ms) and around 300 ms in duration, called the histogram. Here we describe the differences in the histograms recorded during W, SWS, and REM. SWS histograms are always larger than W histograms, and an REM histogram can resemble either. In other words, the optic nerve response to a given stimulus is labile; its configuration depends on whether the rat is asleep or awake. We link this physiological information with the anatomical fact that the brain dorsal raphe region, which is known to have a sleep regulatory role, sends fibers to the rat retina and receives fibers from it. At the cortical electrode, the visual cortical response amplitudes also vary, being largest during SWS. This well known phenomenon often is explained by changes taking place at the thalamic level. However, in the rat, the labile cortical response covaries with the labile optic nerve response, which suggests the cortical response enhancement during SWS is determined more by what happens in the retina than by what happens in the thalamus.

The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.

Temporal distribution of the ganglion cell volleys in the normal rat optic nerve

We describe experiments on behaving rats with electrodes implanted on the cornea, in the optic chiasm, and on the visual cortex; in addition, two red light-emitting diodes (LED) are permanently attached to the skull over the left eye. Recordings timelocked to the LED flashes reveal both the local events at each electrode site and the orderly transfer of visual information from retina to cortex. The major finding is that every stimulus, regardless of its luminance, duration, or the state of retinal light adaptation, elicits an optic nerve volley with a latency of about 10 ms and a duration of about 300 ms. This phenomenon has not been reported previously, so far as we are aware. We conclude that the retina, which originates from the forebrain of the developing embryo, behaves like a typical brain structure: it translates, within a few hundred milliseconds, the chemical information in each pattern of bleached photoreceptors into a corresponding pattern of ganglion cell neuronal information that leaves via the optic nerve. The attributes of each rat ganglion cell appear to include whether the retinal neuropile calls on it to leave after a stimulus and, if so when, within a 300-ms poststimulus epoch. The resulting retinal analysis of the scene, on arrival at the cortical level, is presumed to participate importantly in the creation of visual perceptual experiences.

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Retinal engineering: engrafted neural cell lines locate in appropriate layers.

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Molecular biology of retinal ganglion cells.

Retinal ganglion cells are the output neurons that encode and transmit information from the eye to the brain. Their diverse physiologic and anatomic properties have been intensively studied and appear to account well for a number of psychophysical phenomena such as lateral inhibition and chromatic opponency. In this paper, we summarize our current view of retinal ganglion cell properties and pose a number of questions regarding underlying molecular mechanisms. As an example of one approach to understanding molecular mechanisms, we describe recent work on several POU domain transcription factors that are expressed in subsets of retinal ganglion cells and that appear to be involved in ganglion cell development.

Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development

The Brn-3 subfamily of POU–domain transcription factor genes consists of three highly homologous members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation

We have isolated a dominant mutation, night blindness a (nba), that causes a slow retinal degeneration in zebrafish. Heterozygous nba fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous nba fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting nba fish is not in a photoreceptor cell-specific gene.

The structure and precision of retinal spike trains

Assessing the reliability of neuronal spike trains is fundamental to an understanding of the neural code. We measured the reproducibility of retinal responses to repeated visual stimuli. In both tiger salamander and rabbit, the retinal ganglion cells responded to random flicker with discrete, brief periods of firing. For any given cell, these firing events covered only a small fraction of the total stimulus time, often less than 5%. Firing events were very reproducible from trial to trial: the timing jitter of individual spikes was as low as 1 msec, and the standard deviation in spike count was often less than 0.5 spikes. Comparing the precision of spike timing to that of the spike count showed that the timing of a firing event conveyed several times more visual information than its spike count. This sparseness and precision were general characteristics of ganglion cell responses, maintained over the broad ensemble of stimulus waveforms produced by random flicker, and over a range of contrasts. Thus, the responses of retinal ganglion cells are not properly described by a firing probability that varies continuously with the stimulus. Instead, these neurons elicit discrete firing events that may be the fundamental coding symbols in retinal spike trains.

The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against αvβ3 integrin, but not an anti-β1 antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the αvβ3 integrin on neuronal cells.

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.