Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

Some aspects of amacrine neuron simulation for motion detection

As it is known, there are five types of neurons in the mammalian retinal layer allowing the detection of several important characteristics of the visual image impinging onto the visual system, namely, photoreceptors, horizontal cells, amacrine, bipolar and ganglion cells. And it is a well known fact too, that the amacrine neuron architecture allows a first detection for objects motion, being the most important retinal cell to this function. We have already studied and simulated the Dowling retina model and we have verified that many complex processes in visual detection is performed with the basis of the amacrine cell synaptic connections. This work will show how this structure may be employed for motion detection

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex

Neurons undergoing targeted photolytic cell death degenerate by apoptosis. Clonal, multipotent neural precursor cells were transplanted into regions of adult mouse neocortex undergoing selective degeneration of layer II/III pyramidal neurons via targeted photolysis. These precursors integrated into the regions of selective neuronal death; 15 ± 7% differentiated into neurons with many characteristics of the degenerated pyramidal neurons. They extended axons and dendrites and established afferent synaptic contacts. In intact and kainic acid-lesioned control adult neocortex, transplanted precursors differentiated exclusively into glia. These results suggest that the microenvironmental alterations produced by this synchronous apoptotic neuronal degeneration in adult neocortex induced multipotent neural precursors to undergo neuronal differentiation which ordinarily occurs only during embryonic corticogenesis. Studying the effects of this defined microenvironmental perturbation on the differentiation of clonal neural precursors may facilitate identification of factors involved in commitment and differentiation during normal development. Because photolytic degeneration simulates some mechanisms underlying apoptotic neurodegenerative diseases, these results also suggest the possibility of neural precursor transplantation as a potential cell replacement or molecular support therapy for some diseases of neocortex, even in the adult.

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

N-Methyl-d-aspartate antagonists and apoptotic cell death triggered by head trauma in developing rat brain

Morbidity and mortality from head trauma is highest among children. No animal model mimicking traumatic brain injury in children has yet been established, and the mechanisms of neuronal degeneration after traumatic injury to the developing brain are not understood. In infant rats subjected to percussion head trauma, two types of brain damage could be characterized. The first type or primary damage evolved within 4 hr and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6–24 hr and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus and striatum. Secondary apoptotic damage was more severe than primary excitotoxic damage. Morphometric analysis demonstrated that the N-methyl-d-aspartate receptor antagonists 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate and dizocilpine protected against primary excitotoxic damage but increased severity of secondary apoptotic damage. 2-Sulfo-α-phenyl-N-tert-butyl-nitrone, a free radical scavenger, did not affect primary excitotoxic damage but mitigated apoptotic damage. These observations demonstrate that apoptosis and not excitotoxicity determine neuropathologic outcome after traumatic injury to the developing brain. Whereas free radical scavengers may prove useful in therapy of head trauma in children, N-methyl-d-aspartate antagonists should be avoided because of their propensity to increase severity of apoptotic damage.

A role for the light-dependent phosphorylation of visual arrestin

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cell-specific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.

Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3β (GSK3β) by S9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y216, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y216 phosphorylation on GSK3β in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y216, GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9 phosphorylation and inactivation of GSK3β but did not affect Y216 phosphorylation, suggesting that S9 phosphorylation is sufficient to override GSK3β activation by Y216 phosphorylation. Under the conditions examined, increased Y216 phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y216 phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y216-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y216 phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.

Nontropic actions of neurotrophins: Subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation

Normal aging is associated with a significant reduction in cognitive function across primate species. However, the structural and molecular basis for this age-related decline in neural function has yet to be defined clearly. Extensive cell loss does not occur as a consequence of normal aging in human and nonhuman primate species. More recent studies have demonstrated significant reductions in functional neuronal markers in subcortical brain regions in primates as a consequence of aging, including dopaminergic and cholinergic systems, although corresponding losses in cortical innervation from these neurons have not been investigated. In the present study, we report that aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys (P < 0.001). Further, these age-related reductions are ameliorated by cellular delivery of human nerve growth factor to cholinergic somata in the basal forebrain, restoring levels of cholinergic innervation in the cortex to those of young monkeys (P = 0.89). Thus, (i) aging is associated with a significant reduction in cortical cholinergic innervation; (ii) this reduction is reversible by growth-factor delivery; and (iii) growth factors can remodel axonal terminal fields at a distance, representing a nontropic action of growth factors in modulating adult neuronal structure and function (i.e., administration of growth factors to cholinergic somata significantly increases axon density in terminal fields). These findings are relevant to potential clinical uses of growth factors to treat neurological disorders.

Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: Direct correlation with clinical retinopathy of prematurity

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.