Leber congenital amaurosis (LCA4) has been linked to mutations in the photoreceptor-specific gene Aryl hydrocarbon interacting protein like 1 (Aipl1). To investigate the essential role of AIPL1 in retina, we generated a mouse model of LCA by inactivating the Aipl1 gene. In Aipl1 ؊/؊ retinas, the outer nuclear layer develops normally, but rods and cones then quickly degenerate. Aipl1 ؊/؊ mice have highly disorganized, short, fragmented photoreceptor outer segments and lack both rod and cone electroretinogram responses. Recent biochemical evidence indicates that AIPL1 can enhance protein farnesylation. Our study reveals that rod cGMP phosphodiesterase, a farnesylated protein, is absent and cGMP levels are elevated in AIPL1 ؊/؊ retinas before the onset of degeneration. Our findings demonstrate that AIPL1 enhances the stability of phosphodiesterase and is essential for photoreceptor viability. L eber congenital amaurosis (LCA) has the earliest onset and is the most severe form of inherited retinopathy in humans. LCA is genetically heterogeneous and is generally inherited in an autosomal recessive fashion. LCA is characterized by complete blindness and the near absence of electrical responses to light within 1 year of birth. LCA has been linked to mutations in the gene encoding Aipl1 (1-3). The vast majority of these mutations in Aipl1 are linked to LCA, but a few C-terminal mutations are linked to two other retinal diseases, cone-rod dystrophy and juvenile retinitis pigmentosa (3).AIPL1 is expressed only in retina and pineal gland (1). In adult mouse retina, it is found within the outer plexiform layer and the inner segments of photoreceptors; in humans, AIPL1 is expressed in both developing rods and cones and mature rods (4, 5). Two possibilities for the essential role for AIPL1 in retina have been proposed (4). The first is that AIPL1 enhances an essential farnesylation reaction. Farnesylation is a specific type of prenylation, the addition of a farnesyl or geranylgeranyl residue to specific proteins. Several retinal proteins, cGMP phosphodiesterase (PDE), transducin, and rhodopsin (Rho) kinase (RK) are known to be farnesylated (6-9). Prenylation of retinal proteins is required for maintenance of retinal cytoarchitecture and photoreceptor structure. Inhibition of prenylation causes degeneration of photoreceptor outer segments (10). Prenylation also greatly enhances the stability of cGMP PDE, a protein essential for photoreceptor survival (11). Prenylation of PDE is also necessary for its membrane association (11). These studies suggest that AIPL1 is necessary for the maintenance of photoreceptors. The second possible essential role for AIPL1 is the control of photoreceptor proliferation and or differentiation. AIPL1 interacts with Nedd8-ultimate buster 1 (NUB1), a ubiquitously expressed protein thought to play an important role in regulating cell cycle progression (12). Based on the NUB1 interaction, early expression of AIPL1 in human retina, and the severity of AIPL1 mutations linked to LCA, it has been proposed ...

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca 2؉ -binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact ␣-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg 838 within the dimerization domain establishes the Ca 2؉ sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg 838 substitutions dominantly enhance cGMP synthesis even at the highest Ca 2؉ concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg 838 substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg 838 were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg 838 -substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.

Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such “switch-like” exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments.

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the “Warburg Effect”), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron–glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.

SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membraneintegration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integralmembrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.

Two alleles of an eyeless mutant, chokh (chk), were identified in ongoing zebrafish F(3) mutagenesis screens. Morphologically, chk mutants can be identified at 15 h post-fertilization by the failure of optic primordia to evaginate from the forebrain. The chk phenotype appears specific, as marker genes in the forebrain, midbrain, and pineal are expressed in normal temporal, spatial, and circadian patterns. Sequence analysis of the chk alleles revealed nonsense or missense mutations in the rx3 homeobox. Rx genes encode paired-type homeodomain transcription factors known to be key regulators of eye development in mouse, medaka, Xenopus, and zebrafish. To uncover novel Rx targets, we analyzed the expression of multiple eye development genes in chk. We find that expression of mab21l2, mab21l1 and rx2 are specifically absent in the eye field of chk embryos. Knockdown of Mab21l2 by antisense morpholino microinjections partially phenocopies the rx3 mutation, leading to microphthalmia, incomplete eye maturation, and dramatic increases in apoptotic eye progenitors. We propose that mab21l2 is an early downstream effector of rx3 and is critical for survival of eye progenitors.

Human rods and cones are arranged in a precise spatial mosaic that is critical for optimal functioning of the visual system. However, the molecular processes that underpin specification of cell types within the mosaic are poorly understood. The progressive differentiation of human rods was tracked from fetal week (Fwk) 9 to postnatal (P) 8 months using immunocytochemical markers of key molecules that represent rod progression from post-mitotic precursors to outer segment-bearing functional photoreceptors. We find two phases associated with rod differentiation. The early phase begins in rods on the foveal edge at Fwk 10.5 when rods are first identified, and the rod-specific proteins NRL and NR2e3 are detected. By Fwk 11-12, these rods label for interphotoreceptor retinoid binding protein, recoverin, and aryl hydrocarbon receptor interacting protein-like 1. The second phase occurs over the next month with the appearance of rod opsin at Fwk 15, closely followed by the outer segment proteins rod GTP-gated sodium channel and peripherin. TULP is expressed relatively late at Fwk 18-20 in rods. Each phase proceeds across the retina in a central-peripheral order, such that rods in far peripheral retina are only entering the early phase at the same time that cells in central retina are entering their late phase. During the second half of gestation rods undergo an intracellular reorganization of these proteins, and cellular and OS elongation which continues into infancy. The progression of rod development shown here provides insight into the possible mechanisms underlying human retinal visual dysfunction when there are mutations affecting key rod-related molecules.

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