Correlation between the Serum Concentration of Vitamin A and Disease Severity in Patients Carrying p.G90D in RHO, the Most Frequent Gene Associated with Dominant Retinitis Pigmentosa: Implications for Therapy with Vitamin A.

The pathogenic variant p.G90D in RHO is believed to be responsible for a spectrum of phenotypes, including congenital stationary blindness (for the purpose of this study termed night blindness without degeneration; NBWD), Sector RP, Pericentral RP, and Classic RP. We present a correlation between the serum concentration of vitamin A and disease severity in patients with this variant. This prospective study involved 30 patients from 7 families (17 male; median age 46 years, range 8−73). Full ophthalmological examination including visual acuity, Goldmann perimetry, slit-lamp exam, optical coherence tomography, fundus autofluorescence, and electrophysiology was performed to determine the presenting phenotype. The serum concentration of vitamin A was determined from a fasting blood sample taken on the day of the exam, where it was found that 23.3% (7/30) of patients had NBWD, 13.3% (4/30) had Sector RP, 3.3% (1/30) had Pericentral RP, and 60% (18/30) had Classic RP. Multiple logistic regression revealed a significantly higher probability of having a milder phenotype (NBWD or Sector RP) in association with younger age (p < 0.05) and a higher concentration of vitamin A (p < 0.05). We hypothesize that vitamin A in its 11-cis-retinal form plays a role in stabilizing the constitutively active p.G90D rhodopsin and its supplementation could be a potential treatment strategy for p.G90D RHO patients.

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes.

Inherited retinal diseases (IRDs) have been in the front line of gene therapy development for the last decade, providing a useful platform to test novel therapeutic approaches. More than 40 clinical trials have been completed or are ongoing, tackling autosomal recessive and X-linked conditions, mostly through adeno-associated viral vector delivery of a normal copy of the disease-causing gene. However, only recently has autosomal dominant (ad) disease been targeted, with the commencement of a trial for rhodopsin (RHO)-associated retinitis pigmentosa (RP), implementing antisense oligonucleotide (AON) therapy, with promising preliminary results (NCT04123626).Autosomal dominant RP represents 15%-25% of all RP, with RHO accounting for 20%-30% of these cases. Autosomal dominant macular and cone-rod dystrophies (MD/CORD) correspond to approximately 7.5% of all IRDs, and approximately 35% of all MD/CORD cases, with the main causative gene being BEST1 Autosomal dominant IRDs are not only less frequent than recessive, but also tend to be less severe and have later onset; for example, an individual with RHO-adRP would typically become severely visually impaired at an age 2-3 times older than in X-linked RPGR-RP.Gain-of-function and dominant negative aetiologies are frequently seen in the prevalent adRP genes RHO, RP1 and PRPF31 among others, which would not be effectively addressed by gene supplementation alone and need creative, novel approaches. Zinc fingers, RNA interference, AON, translational read-through therapy, and gene editing by clustered regularly interspaced short palindromic repeats/Cas are some of the strategies that are currently under investigation and will be discussed here.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Developmental effects of constant light on circadian behaviour and gene expressions in zebra finches: Insights into mechanisms of metabolic adaptation to aperiodic environment in diurnal animals.

A most crucial feature of biological adaptation is the maintenance of a close temporal relationship of behaviour and physiology with prevailing 24-h light-dark environment, which is rapidly changing with increasing nighttime illumination. This study investigated developmental effects of the loss of night on circadian behaviour, metabolism and gene expressions in diurnal zebra finches born and raised under LL, with controls on 12L:12D. Birds under LD were entrained, and showed normal body mass and a significant 24-h rhythm in both activity-rest pattern and mRNA expression of candidate genes that we measured. But, under LL, birds gained weight and accumulated lipid in the liver. Intriguingly, at the end of the experiment, the majority (4/5th) of birds under LL were rhythmic in activity despite arrhythmic expression in the hypothalamus of c-Fos (neuronal activity), Rhodopsin and Mel1-a genes (light perception), and clock genes (Bmal1, Per2 and Rev-erb ß). In peripheral tissues, LL induced variable clock gene expressions. Whereas 24-h mRNA rhythm was abolished for Bmal1 in both liver and gut, it persisted for Per2 and Rev-erb ß in liver, and for Per2 in gut. Further, we found under LL, the loss of 24-h rhythm in hepatic expression of Fasn and Cd36/Fat (biosynthesis and its uptake), and gut expression of Sglt1, Glut5, Cd36 and Pept1 (nutrient absorption) genes. As compared to LD, baseline mRNA levels of Fasn and Cd36 genes were attenuated under LL. Among major transporter genes, Sglt1 (glucose) and Cd36 (fat) genes were arrhythmic, while Glut5 (glucose) and Pept1 (protein) genes were rhythmic but with phase differences under LL, compared to LD. These results demonstrate dissociation of circadian behaviour from clock gene rhythms, and provide molecular insights into possible mechanisms at different levels (behaviour and physiology) that diurnal animals might employ in order to adapt to an emerging overly illuminated-night urban environment.

Noninvasive Electrical Stimulation Improves Photoreceptor Survival and Retinal Function in Mice with Inherited Photoreceptor Degeneration.

Purpose: Neurons carry electrical signals and communicate via electrical activities. The therapeutic potential of electrical stimulation (ES) for the nervous system, including the retina, through improvement of cell survival and function has been noted. Here we investigated the neuroprotective and regenerative potential of ES in a mouse model of inherited retinal degeneration. Methods: Rhodopsin-deficient (Rho-/-) mice received one or two sessions of transpalpebral ES or sham treatments for 7 consecutive days. Intraperitoneal injection of 5-ethynyl-2'-deoxyuridine was used to label proliferating cells. Weekly electroretinograms were performed to monitor retinal function. Retinal morphology, photoreceptor survival, and regeneration were evaluated in vivo using immunohistochemistry and genetic fate-mapping techniques. Müller cell (MC) cultures were employed to further define the optimal conditions of ES application. Results: Noninvasive transpalpebral ES in Rho-/- mice improved photoreceptor survival and electroretinography function in vivo. ES also triggered residential retinal progenitor-like cells such as MCs to reenter the cell cycle, possibly producing new photoreceptors, as shown by immunohistochemistry and genetic fate-mapping techniques. ES directly stimulated cell proliferation and the expression of progenitor cell markers in MC cultures, at least partially through bFGF signaling. Conclusions: Our study showed that transpalpebral ES improved photoreceptor survival and retinal function and induced the proliferation, probably photoreceptor regeneration, of MCs; this occurs via stimulation of the bFGF pathways. These results suggest the exciting possibility of applying noninvasive ES as a versatile tool for preventing photoreceptor loss and mobilizing endogenous progenitors for reversing vision loss in patients with photoreceptor degeneration.

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease.

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

Filtration of Short-Wavelength Light Provides Therapeutic Benefit in Retinitis Pigmentosa Caused by a Common Rhodopsin Mutation.

Purpose: The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit. Methods: Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts. Results: Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group. Conclusions: Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

Docosahexaenoic acid promotes differentiation of photoreceptor cells in three-dimensional neural retinas.

Retinal tissues generated from human pluripotent stem cells can be an excellent tool for investigating pathogenesis of retinal diseases and developing new pharmacologic therapies. Moreover, patient derived retinal tissues could allow for retinal transplantation therapy for degenerative retinal diseases. However, obtaining retinal tissues with matured photoreceptor outer segments, which are essential for photoreceptor functions, is currently challenging. Here we investigated the effects of docosahexaenoic acid (DHA) for maturation of photoreceptor outer segments at the late stage and visual chromophore analog, 9-cis-retinal for the early stage of differentiation to three-dimensional (3D)-retinal tissues from human embryonic stem cells (hESCs), respectively. In the presence of DHA, differentiated 3D-retinal tissues demonstrated improved maturation of photoreceptor outer segments and increased number of photoreceptor cells compared with tissues without DHA. Increased mRNA expression of mature photoreceptor markers was additionally documented in retinal tissues cultured with DHA. Conversely supplementation with 9-cis-retinal failed to improve differentiation of retinal tissues perhaps due to chronic aldehyde toxicity. The current study demonstrated that the addition of DHA to culture medium can help promote differentiation of photoreceptor outer segments in vitro and utilization of this methodology may lead to future therapies for patients with blinding diseases.

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

Neuromodulatory Regulation of Behavioral Individuality in Zebrafish.

Inter-individual behavioral variation is thought to increase fitness and aid adaptation to environmental change, but the underlying mechanisms are poorly understood. We find that variation between individuals in neuromodulatory input contributes to individuality in short-term habituation of the zebrafish (Danio Rerio) acoustic startle response (ASR). ASR habituation varies greatly between individuals, but differences are stable over days and are heritable. Acoustic stimuli that activate ASR-command Mauthner cells also activate dorsal raphe nucleus (DRN) serotonergic neurons, which project to the vicinity of the Mauthner cells and their inputs. DRN neuron activity decreases during habituation in proportion to habituation and a genetic manipulation that reduces serotonin content in DRN neurons increases habituation, whereas serotonergic agonism or DRN activation with ChR2 reduces habituation. Finally, level of rundown of DRN activity co-segregates with extent of behavioral habituation across generations. Thus, variation between individuals in neuromodulatory input contributes to individuality in a core adaptive behavior. VIDEO ABSTRACT.

A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.

PURPOSE: Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS: Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS: Two stabilization assays of P23H mutant opsin were developed and validated, one based on ß-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS: Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

Optogenetics-enabled dynamic modulation of action potential duration in atrial tissue: feasibility of a novel therapeutic approach.

AIMS: Diseases that abbreviate the cardiac action potential (AP) by increasing the strength of repolarizing transmembrane currents are highly arrhythmogenic. It has been proposed that optogenetic tools could be used to restore normal AP duration (APD) in the heart under such disease conditions. This study aims to evaluate the efficacy of an optogenetic treatment modality for prolonging pathologically shortened APs in a detailed computational model of short QT syndrome (SQTS) in the human atria, and compare it to drug treatment. METHODS AND RESULTS: We used a human atrial myocyte model with faster repolarization caused by SQTS; light sensitivity was inscribed via the presence of channelrhodopsin-2 (ChR2). We conducted simulations in single cells and in a magnetic resonance imaging-based model of the human left atrium (LA). Application of an appropriate optical stimulus to a diseased cell dynamically increased APD, producing an excellent match to control AP (<1.5 mV deviation); treatment of a diseased cell with an AP-prolonging drug (chloroquine) also increased APD, but the match to control AP was worse (>5 mV deviation). Under idealized conditions in the LA (uniform ChR2-expressing cell distribution, no light attenuation), optogenetics-based therapy outperformed chloroquine treatment (APD increased to 87% and 81% of control). However, when non-uniform ChR2-expressing cell distribution and light attenuation were incorporated, optogenetics-based treatment was less effective (APD only increased to 55%). CONCLUSION: This study demonstrates proof of concept for optogenetics-based treatment of diseases that alter atrial AP shape. We identified key practical obstacles intrinsic to the optogenetic approach that must be overcome before such treatments can be realized.

Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds. RESULTS: We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient's iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient's iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes. CONCLUSION: The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.

Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms.

BACKGROUND: Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes. RESULTS: Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the ß4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naive mice and activated nonsomatostatin neurons in the IPN. CONCLUSIONS: Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Retinitis pigmentosa mutants provide insight into the role of the N-terminal cap in rhodopsin folding, structure, and function.

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.

Vitamin A supplementation ameliorates obesity-associated retinal degeneration in WNIN/Ob rats.

OBJECTIVE: Obesity is associated with various health afflictions, including ocular complications such as diabetic retinopathy, high intraocular pressure, cataracts, and macular degeneration. We previously reported progressive retinal degeneration after the onset of obesity in the spontaneously obese rat (WNIN/Ob) model. In the present study, we investigated vitamin A supplementation to ameliorate obesity-associated retinal degeneration in the WNIN/Ob rat. METHODS: Five-month-old male WNIN/Ob obese (O) and lean (L) control rats were fed with vitamin A 2.6 mg (L/O-I), 26 mg (L/O-II), 52 mg (L/O-III), and 129 mg (L/O-IV) per kilogram of diet as retinyl palmitate for 4 mo 2 wk. Retinal morphology and retinal gene expression were assessed by histologic, immunohistochemical, and real-time polymerase chain reaction methods. RESULTS: Supplementation of vitamin A at 26 or 52 mg significantly modulated the expression of retinal genes in the O but not in the L phenotype. Vitamin A supplementation significantly upregulated the expression of genes, such as rhodopsin, rod arrestin, phosphodiesterase, transducins, and fatty acid elongase-4, that were otherwise downregulated in O rat retina. The expression of glial fibrillary acidic protein was downregulated by vitamin A feeding in O rat retina. The immunohistochemical and histologic findings corroborated the gene expression data. The effects were significant at a 26- or 52-mg dose of vitamin A. CONCLUSION: Vitamin A supplementation alleviated obesity-associated retinal degeneration in the WNIN/Ob rat.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.