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.

Mutations in the gene coding for AIPL1 cause Leber congenital amaurosis (LCA), a severe form of childhood blindness. The severity in disease is reflected in the complete loss of vision and rapid photoreceptor degeneration in the retinas of mice deficient in AIPL1. Our previous observations suggest that rod photoreceptor degeneration in retinas lacking AIPL1 is due to the massive reduction in levels of rod cGMP phosphodiesterase (PDE6) subunits (␣, ␤, and ␥). To date, the crucial link between AIPL1 and the stability of PDE6 subunits is not known. In this study using ex vivo pulse label analysis, we demonstrate that AIPL1 is not involved in the synthesis of PDE6 subunits. However, ex vivo pulse-chase analysis clearly shows that in the absence of AIPL1, rod PDE6 subunits are rapidly degraded by proteasomes. We further demonstrate that this rapid degradation of PDE6 is due to the essential role of AIPL1 in the proper assembly of synthesized individual PDE6 subunits. In addition, using a novel monoclonal antibody generated against AIPL1, we show that the catalytic subunit (␣) of PDE6 associates with AIPL1 in retinal extracts. Our studies establish that AIPL1 interacts with the catalytic subunit (␣) of PDE6 and is needed for the proper assembly of functional rod PDE6 subunits. Leber congenital amaurosis (LCA)2 is an early childhood blinding disease that affects the retina. At a very young age, children affected with LCA lack both scotopic and photopic visual response implying both rod and cone photoreceptors dysfunction (1). To date, mutations in 14 genes, including the Aipl1 gene have been linked to this disease (1, 2). A recent study showed that an adult LCA patient with a mutation in Aipl1 has no visual response (3). In addition, immunohistochemistry using rod markers showed that the patient had no rod photoreceptors. Although some remnants of cone photoreceptors remained, the outer segments were completely missing, suggesting a degeneration of both rod and cone photoreceptors in this LCA patient.AIPL1 is specifically expressed in retina and in the pineal gland. In retina, AIPL1 is expressed in young and adult rod photoreceptors (4, 5). However, AIPL1 is expressed in cones transiently and is thought to be absent from adult cones (3). Two independent studies demonstrated that mice lacking AIPL1 undergo a rapid and severe retinal degeneration with both rod and cone photoreceptor loss (6, 7). Although AIPL1 was not essential for the initial formation of photoreceptor cells, the photoreceptor cells were not functional, as they could not evoke any light-dependent electrical response (6). These results are consistent with the disease characteristics of LCA (8). Our previous studies show that before degeneration, rod PDE6 activity and the levels of PDE6 protein are drastically reduced in the absence of AIPL1 (6). In addition to the knockout mouse, an AIPL1 knockdown mouse was created in which AIPL1 expression was reduced to 20 -25% of wild-type levels (9). The knockdown mouse showed normal development and retinal morpholo...

We have determined the crystal structure of a novel regulatory protein (MGP-40) from the mammary gland. This protein is implicated as a protective signaling factor that determines which cells are to survive the drastic tissue remodeling that occurs during involution. It has been indicated that certain cancers could surreptitiously utilize the proposed normal protective signaling by proteins of this family to extend their own survival and thereby allow them to invade the organ and metastasize. In view of this, MGP-40 could form an important target for rational structure-based drug design against breast cancer. It is a single chain, glycosylated protein with a molecular mass of 40 kDa. It was isolated from goat dry secretions and has been cloned and sequenced. It was crystallized by microdialysis from 20 mg ml Mammary glands secrete a class of very important proteins during involution. We have isolated a glycoprotein from goat dry secretions which has a molecular mass of 40 kDa. This mammary gland protein has been named MGP-40.

Leber congenital amaurosis (LCA) caused by mutations in Aryl hydrocarbon receptor interacting protein like-1 (Aipl1) is a severe form of childhood blindness. At 4 weeks of age, a mouse model of LCA lacking AIPL1 exhibits complete degeneration of both rod and cone photoreceptors. Rod cell death occurs due to rapid destabilization of rod phosphodiesterase, an enzyme essential for rod survival and function. However, little is understood regarding the role of AIPL1 in cone photoreceptors. Cone degeneration observed in the absence of AIPL1 could be due to an indirect 'bystander effect' caused by rod photoreceptor death or a direct role for AIPL1 in cones. To understand the importance of AIPL1 in cone photoreceptor cells, we transgenically expressed hAIPL1 exclusively in the rod photoreceptors of the Aipl1(-/-) mouse. Transgenic expression of hAIPL1 restored rod morphology and the rod-derived electroretinogram response, but cone photoreceptors were non-functional in the absence of AIPL1. In addition, the cone photoreceptors degenerate, but at a slower rate compared with Aipl1(-/-) mice. This degeneration is linked to the highly reduced levels of cone PDE6 observed in the hAIPL1 transgenic mice. Our studies demonstrate that AIPL1 is needed for the proper functioning and survival of cone photoreceptors. However, rod photoreceptors also provide support that partially preserves cone photoreceptors from rapid death in the absence of AIPL1.

Bardet-Biedl syndrome (BBS) is a genetic disorder affecting multiple systems and organs in the body. Several mutations in genes associated with BBS affect only photoreceptor cells and cause nonsyndromic retinitis pigmentosa (RP), raising the issue of why certain mutations manifest as a systemic disorder whereas other changes in the same gene affect only a specific cell type. Here, we show that cell-type-specific alternative splicing is responsible for confining the phenotype of the A-to-G substitution in the 3= splice site of BBS8 exon 2A (IVS1-2A>G mutation) in the BBS8 gene to photoreceptor cells. The IVS1-2A>G mutation leads to missplicing of BBS8 exon 2A, producing a frameshift in the BBS8 reading frame and thus eliminating the protein specifically in photoreceptor cells. Cell types other than photoreceptors skip exon 2A from the mature BBS8 transcript, which renders them immune to the mutation. We also show that the splicing of Bbs8 exon 2A in photoreceptors is directed exclusively by redundant splicing enhancers located in the adjacent introns. These intronic sequences are sufficient for photoreceptor-cell-specific splicing of heterologous exons, including an exon with a randomized sequence. The BBSome is a multiprotein complex that is thought be required for the transport of proteins in and out of the cilia. The BBSome interacts with intraflagellar transport A (IFT-A) and IFT-B complexes and promotes the assembly of the IFT machinery (1-7). Several proteins, such as the G-protein-coupled receptors Smo, Sstr3, Mchr1, and Vipr2, depend on the BBSome for their ciliary transport, suggesting a possible role for the BBSome as an adapter that connects the IFT complex to its cargo in primary cilia (2,8,9). Mutations in genes that encode BBSome components and proteins associated with the BBSome are linked to systemic Bardet-Biedl syndrome (BBS). BBS is an autosomal recessive ciliopathy caused by defects in the BBSome that disrupt the normal ciliary function throughout the body. BBS symptoms include retinitis pigmentosa (RP), skeletal malformations, mental retardation, obesity, hearing impairment, shortened limbs, polydactyly, and kidney cysts (10, 11). In photoreceptor cells, BBSome deficiency leads to defects in rod outer-segment formation and localization of rod opsin and, ultimately, photoreceptor cell death (10-13). The severity of the BBS symptoms can vary considerably due to the nature of the mutation and the genetic background. Interestingly, phenotypes of different mutations in the same gene can range from a classical BBS that affects multiple systems to nonsyndromic RP, where the phenotype is limited to loss of photoreceptor function. For example, the ARL6 (BBS3) A89V, BBS1 M390R, and BBS8 IVS1-2AϾG mutations cause nonsyndromic RP, while several other mutations in the same genes manifest as classical BBS, presenting additional symptoms such as obesity, hearing impairment, polydactyly, and mental retardation in addition to the loss of vision (7,(14)(15)(16)(17)(18)(19). The existence of BBSome mutations th...

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca 2+ current, suggesting interactions of transducin with the synaptic machinery.retina | adaptation | presynaptic modulation | SNARE complex | palmitoylation

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Prenylation is the posttranslational modification of a carboxylterminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.protein posttranslational modification | protein transport | retinal degeneration | methyl esterification P osttranslational modifications increase a protein's functional repertoire, regulating protein-protein interactions and targeting to cellular components. One such posttranslational modification is prenylation, a three-step process that first adds a farnesyl or geranylgeranyl lipid to the C-terminal cysteine of proteins ending in a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid residue) (1-3). The next step is proteolysis of the last three amino acids (-AAX) by the protease RAS-converting enzyme 1 (RCE1) or zinc metalloproteinase sterile-24 homologue (ZMPSTE24) (4, 5). The final step is the methyl esterification of the newly exposed isoprenylcysteine residue catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT) (4).Prenylation enables peripheral membrane proteins to interact with membranes and facilitates their interaction with protein partners (2). The significance of the proteolysis and methylation steps is not entirely clear (4). In general, farnesylated proteins require postprenylation processing for proper localization, whereas the more hydrophobic geranylgeranylated proteins do not (6). Germline knockout of Rce1 or Icmt results in embryonic lethality in mice, demonstrating the importance of these processing steps during development (7,8). Subsequent conditional knockout studies revealed that proteolysis is crucial in the heart but is dispensable in liver and bone marrow (9, 10). Although Rce1 is expressed in the brain and eye, the role of RCE1-mediated endoproteolysis in neuronal tissue in vivo is not ...