Restoration of vision after transplantation of photoreceptors

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1?/? mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1?/? mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.     

Primary structure of the alpha-subunit of transducin and its relationship to ras proteins

A group of membrane-associated guanine nucleotide binding proteins (G-proteins) are essential for transducing signals generated at cell-surface receptors into changes in cellular function and metabolism. These proteins are a complex of three subunits designated alpha, beta and gamma. The alpha-subunit is responsible for binding guanine nucleotides and seems to be characteristic of each protein. Transducin, a member of this protein family, mediates visual transduction by coupling the signal of photolysed rhodopsin with activation of a cyclic GMP phosphodiesterase. We have now cloned and sequenced the complementary DNA encoding the alpha-subunit of bovine retinal transducin and from this we have deduced the complete amino-acid sequence. The transducin alpha-subunit shares several homologous amino-acid sequences with ras gene products. The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit.     

A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas

Rhodopsin is a visual pigment ubiquitous in multicellular animals. If visual pigments have a common ancient origin, as is believed, then some unicellular organisms might also use a rhodopsin photoreceptor. We show here that the unicellular alga Chlamydomonas does indeed use a rhodopsin photoreceptor. We incorporated analogues of its retinal chromophore into a blind mutant; normal photobehaviour was restored and the colour of maximum sensitivity was shifted in a manner consistent with the nature of the retinal analogue added. The data suggest that 11-cis-retinal is the natural chromophore and that the protein environment of this retinal is similar to that found in bovine rhodopsin, suggesting homology with the rhodopsins of higher organisms. This is the first demonstration of a rhodopsin photoreceptor in an alga or eukaryotic protist and also the first report of behavioural spectral shifts caused by exogenous synthetic retinals in a eukaryote. A survey of the morphology and action spectra of other protists suggests that rhodopsins may be common photoreceptors of chlorophycean, prasinophycean and dinophycean algae. Thus, Chlamydomonas represents a useful new model for studying photoreceptor cells.     

A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa.

The group of retinopathies termed retinitis pigmentosa (RP) greatly contribute to visual dysfunction in man with a frequency of roughly 1 in 4,000. We mapped the first autosomal dominant RP (adRP) gene to chromosome 3q, close to the gene encoding rhodopsin, a rod photoreceptor pigment protein. Subsequently, mutations in this gene have been implicated as responsible for some forms of adRP. Another adRP gene has been mapped to chromosome 8p. A third adRP gene in a large Irish pedigree has been mapped to chromosome 6p, showing tight linkage with the gene for peripherin, a photoreceptor cell-specific glycoprotein, which is thus a strong candidate for the defective gene. We have now identified a three-base-pair deletion which results in the loss of one of a pair of highly conserved cysteine residues in the predicted third transmembrane domain of peripherin. This deletion segregates with the disease phenotype but is not present in unaffected controls, and suggests that mutant peripherin gives rise to retinitis pigmentosa.     

Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa.

The murine retinal degeneration slow (rds) gene is a semidominant mutation with a phenotype having rod and cone photoreceptors that develop abnormally and then slowly degenerate. The phenotype is a possible model for retinitis pigmentosa, one of the scores of hereditary human retinal degenerations, which is also characterized by photoreceptor degeneration. We report here three mutations of the human homologue of the rds gene (RDS) that cosegregate with autosomal dominant retinitis pigmentosa in separate families. Our results indicate that some cases of autosomal dominant retinitis pigmentosa are due to mutations at the RDS locus.     

Subsecond deactivation of transducin by endogenous GTP hydrolysis

The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.     

Multiple mRNA species with distinct 3' termini are transcribed from the beta 2-microglobulin gene

beta 2-Microglobulin is the small, relatively invariant subunit of a family of cell-surface glycoproteins encoded within the major histocompatibility complex (MHC). Proteins associated with beta 2-microglobulin in the mouse include the classical transplantation antigens (H-2K, D and L), the thymus leukaemia antigen (TL) and certain haematopoietic cell differentiation antigens (Qa-1 and Qa-2). The genes encoding these proteins are members of a large, multigene family. In contrast, beta 2-microglobulin is encoded by a single copy gene on mouse chromosome 2 (refs 5, 6). We have shown that this gene consists of four coding blocks separated by three intervening sequences. We now demonstrate that the single beta 2-microglobulin gene is transcribed into at least two different size classes of mRNA that differ in the lengths of their 3' untranslated regions. We further show that three polyadenylation signals and a poly (A) tail are encoded at the 3' end of the gene.     

Photoreceptor-specific degeneration caused by tunicamycin

The antibiotic tunicamycin inhibits the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyisoprenol, a key intermediate in the formation of the asparagine-linked oligosaccharides of glycoproteins. The effects of tunicamycin have been studied in various biological systems, primarily with the aim of elucidating the role of the carbohydrate moieties in the cellular function of glycoproteins. Rhodopsin, the visual pigment of retinal rod photoreceptor cells, is a membrane glycoprotein which consists of a single polypeptide chain (opsin) to which a chromophoric prosthetic group (II-cis-retinaldehyde) and two asparagine-linked oligosaccharide chains are covalently attached. The glycosylation of opsin can be blocked with tunicamycin in vitro in conditions where polypeptide synthesis is only slightly decreased. We have reported that tunicamycin can disrupt the normal assembly of rod outer segment membranes in vitro without significantly inhibiting the biosynthesis or intracellular transport of opsin. Here we report that intraocular injection of tunicamycin produces a photoreceptor-specific degeneration characterized by progressive shortening of rod outer segment, decreased membrane assembly, and eventual photoreceptor cell death.     

Signal clipping by the rod output synapse

The properties of synapses between retinal neurons make an essential contribution to early visual processing. Light produces a graded hyperpolarization in photoreceptors, up to 25 mV in amplitude, and it is conventionally assumed that all of this response range is available for coding visual information. We report here, however, that the rod output synapse rectifies strongly, so that only potential changes within 5 mV of the rod dark potential are transmitted effectively to postsynaptic horizontal cells. This finding is consistent with the voltage-dependence of the calcium current presumed to control neurotransmitter release from rods. It suggests functional roles for the strong electrical coupling of adjacent rods and the weak electrical coupling of adjacent rods and cones. The existence of photoreceptor coupling resolves the apparent paradox that rods have a 25 mV response range, while signals greater than 5 mV in amplitude are clipped during synaptic transmission. We predict that the strengths of rod-rod and rod-cone coupling are quantitatively linked to the relationship between the rod response range and the synapse operating range.     

Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP.

The photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase gamma-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its gamma-subunits, accelerates GTPase activity by several fold. Thus the gamma-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase alpha and beta-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

An Fc receptor structurally related to MHC class I antigens

Maternal immunoglobulin G transmitted to the fetus or newborn provides humoral immunity for the first weeks of mammalian life. Fc receptors on intestinal epithelial cells of the neonatal rat (FcRn) mediate the uptake of IgG from milk. Affinity-purified FcRn is resolved by SDS-PAGE into components of relative molecular masses 45,000-53,000 (p51) and about 14,000 (p14). We report the identification of the smaller component as beta 2-microglobulin. Association of beta 2-microglobulin with p51 was confirmed by crosslinking in intestinal epithelial cell brush borders. We have cloned a cDNA encoding the presumptive Fc-binding subunit, p51, and its predicted primary structure has three extracellular domains and a transmembrane region which are all homologous to the corresponding domains of class I major histocompatibility complex (MHC) antigens. This is the first time a function has been assigned to an MHC antigen-related molecule.     

Complete structure and expression in transfected cells of high affinity IgE receptor

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.     

Complexity of human T-cell antigen receptor beta-chain constant- and variable-region genes

Immune systems of vertebrates function via two types of effector cells, B and T cells, which are capable of antigen-specific recognition. The immunoglobulins, which serve as antigen receptors on B cells, have been well characterized with respect to gene structure, unlike the T-cell receptors. Recently, cDNA clones thought to correspond to the beta-chain locus of the human and mouse T-cell receptor have been described. The presumptive beta-chain clones detect gene rearrangement specifically in T-cell DNA and show homology with immunoglobulin light chains. The similarity of the T-cell beta-chain gene system to the immunoglobulin genes has been further demonstrated by the recent observation of variable- and constant-region gene segments as well as joining segments and putative diversity segments. We report here the characterization of cDNA and genomic clones encoding human T-cell receptor beta-chain genes. There are two constant-region genes (C beta 1 and C beta 2), each capable of rearrangement and expression as RNA. The gene arrangement, analogous to that of mouse beta-chain genes, shows strong evolutionary conservation of the dual C beta gene system in these two species.     

GABA and GAD immunoreactivity of photoreceptor terminals in primate retina

Within the vertebrate retina, two types of photoreceptor cells--the rods and cones--transduce visual signals and convey this information through synapses with bipolar and horizontal cells. Although the neurotransmitter at these first-order synapses has not been identified, electrophysiological studies suggest that it might be excitatory. In the present study, however, we have found photoreceptor terminals in the rhesus monkey retina which are immunoreactive with antibodies to either gamma-aminobutyric acid (GABA) or L-glutamic acid decarboxylase (GAD, an enzyme involved in the synthesis of GABA). In the perifoveal region of the retina, approximately 25% of presynaptic profiles having ultrastructural characteristics of either rod or cone terminals are immunoreactive with one or the other antibody. This evidence for a putatively inhibitory neurotransmitter in photoreceptor terminals challenges present understanding of retinal synaptic function.     

Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.     

Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods

It is generally accepted that the light response in retinal rods involves a reduction of ionic permeability (predominantly to Na+) in the plasma membrane of the outer segment and that this is mediated by an internal messenger which diffuses between the disk and plasma membranes. There is controversy, however, over the identity of the diffusible substance; two alternative schemes have received widespread support (for review see refs 1,2). According to the 'calcium hypothesis', light stimulates the release into the cytoplasm of calcium, leading to the blockage of channels which are normally open in darkness, whereas based on the 'cyclic nucleotide hypothesis', cyclic GMP causes the opening of channels in the dark, but is hydrolysed by a light-activated phosphodiesterase. We report here effects of introducing calcium buffers and cyclic GMP into the rod cytoplasm by means of a patch pipette, which seem to be inconsistent with the calcium hypothesis.     

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.