G-protein betagamma-complex is crucial for efficient signal amplification in vision

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. While much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary βγ-complex in this process remains a mystery. Here we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin βγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions

Alteration of Retinal Rod Outer Segment Membrane Fluidity in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol (Chol) biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol and decreased Chol levels. Progressive retinal degeneration occurs in an animal model of SLOS, induced by treating rats with AY9944, a selective inhibitor of the enzyme affected in SLOS. Here we evaluated alterations in the biochemical and physical properties of retinal rod outer segment (ROS) membranes in this animal model. At 1 month of AY9944 treatment, there were modest alterations in fatty acid composition, but no significant differences in cis-parinaric acid (cPA) spectroscopic parameters in ROS membranes from treated versus control rats. However, at 3 months, ROS docosahexaenoic acid (DHA) content was dramatically reduced, and cPA fluorescence anisotropy values were decreased, relative to controls. Also, 1, 6-diphenyl-1, 3, 5-hexatriene exhibited decreased rotational motion and increased orientational order in ROS membranes from 3 month-old AY9944-treated rats, relative to controls. No significant changes in protein:lipid ratios were observed; however, rhodopsin regenerability was compromised by 3 months of treatment. These findings are consistent with reduced ROS membrane fluidity in the SLOS rat model, relative to controls, primarily due to the dramatic reduction inmembraneDHA levels, rather than altered sterol composition

Retention of function without normal disc morphogenesis occurs in cone but not rod photoreceptors

It is commonly assumed that photoreceptor (PR) outer segment (OS) morphogenesis is reliant upon the presence of peripherin/rds, hereafter termed Rds. In this study, we demonstrate a differential requirement of Rds during rod and cone OS morphogenesis. In the absence of this PR-specific protein, rods do not form OSs and enter apoptosis, whereas cone PRs develop atypical OSs and are viable. Such OSs consist of dysmorphic membranous structures devoid of lamellae. These tubular OSs lack any stacked lamellae and have reduced phototransduction efficiency. The loss of Rds only appears to affect the shape of the OS, as the inner segment and connecting cilium remain intact. Furthermore, these structures fail to associate with the specialized extracellular matrix that surrounds cones, suggesting that Rds itself or normal OS formation is required for this interaction. This study provides novel insight into the distinct role of Rds in the OS development of rods and cones

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of ProteinN-Glycosylation

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step indolichol synthesis. Recessive mutations inDHDDScause retinitis pigmentosa(RP59), resulting in blindness. We hypothesized that rod photoreceptor-specificablation ofDhddswould cause retinal degeneration due to diminished dolichol-dependent proteinN-glycosylation.Dhddsflx/flxmice were crossed with rod-spe-cific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specificDhddsknockout mice (Dhddsflx/flxiCre+).In vivomorphological and electrophys-iological evaluation ofDhddsflx/flxiCre+retinas revealed mild retinal dysfunctionat postnatal (PN) 4 weeks, compared with age-matched controls; however, rapidphotoreceptor degeneration ensued, resulting in almost complete loss of rodsand cones by PN 6 weeks. Retina dolichol levels were markedly decreased byPN 4 weeks inDhddsflx/flxiCre+mice, relative to controls; despite this,N-glycosyl-ation of retinal proteins, including opsin (the dominant rod-specific glycoprotein),persisted inDhddsflx/flxiCre+mice. These findings challenge the conventionalmechanistic view of RP59 as a congenital disorder of glycosylation

Robust Lysosomal Calcium Signaling Through Channel TRPML1 is Impaired by Lysosomal Lipid Accumulation

The transient receptor potential cation channelmucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calciuminRPE cells andwhether such release is affectedby lysosomal accumulations.The TRPM LagonistML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-L-phenylalanine-b-naphthylamide. Treatment with desipramine toinhibit acidsphingomyelinase orYM201636 to inhibitPIKfyve also reducedthe cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML-/- mice showed no response to ML-SA1. Cotreatmentwith chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomalCa2+ release.LysosomalCa2+ releasewas also impaired in RPEcells from the ATP-binding cassette, subfamily A, member 4-/-mouse model of Stargardt\u27s retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models,suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calciumlevels in RPE cells, but this response is reduced by lysosomal accumulation.-Gomez, N. M.,Lu,W.Lim, J. C.,Kiselyov, K.,Campagno, K.E.,Grishchuk,Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation. FASEB J. 32, 782-794 (2018). www.fasebj.org. © FASEB

Compromised Phagosome Maturation Underlies RPE Pathology in Cell Culture and Whole Animal Models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group

Differential Developmental Deficits in Retinal Function in the Absence of either Protein Tyrosine Sulfotransferase-1 or -2

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1−/− and Tpst2−/− mice, retinal function was compromised during early development. However, Tpst1−/− retinas became electrophysiologically normal by postnatal day 90 while Tpst2−/− mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina

A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds+/− mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^(–/–)LacZ and Abcg4^(–/–)LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^(–/–) mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior

Oxysterols and Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome: Implications for an Improved Therapeutic Intervention

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive human disease caused by mutations in the gene encoding 7-dehydrocholesterol (7DHC) reductase (DHCR7), resulting in abnormal accumulation of 7DHC and reduced levels of cholesterol in bodily tissues and fluids. A rat model of the disease has been created by treating normal rats with the DHCR7 inhibitor, AY9944, which causes progressive, irreversible retinal degeneration. Herein, we review the features of this disease model and the evidence linking 7DHC-derived oxysterols to the pathobiology of the disease, with particular emphasis on the associated retinal degeneration. A recent study has shown that treating the rat model with cholesterol plus suitable antioxidants completely prevents the retinal degeneration. These findings are discussed with regard to their translational implications for developing an improved therapeutic intervention for SLOS over the current standard of care