Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.

A Mixture of U.S. Food and Drug Administration-Approved Monoaminergic Drugs Protects the Retina From Light Damage in Diverse Models of Night Blindness.

Purpose: The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods: Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (ß1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results: The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions: Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.

Analysis of aging-dependent changes in taste sensitivities of the senescence-accelerated mouse SAMP1.

To investigate aging-dependent changes in taste sensitivities, we performed behavioral tests regarding taste sensitivity among young and old SAMP1 mice. In this senescence-accelerated mice model, dramatic changes in taste sensitivities were observed at least 70 weeks old. As for in a brief access test, old mice showed significantly increased taste sensitivity to bitter, salty, sweet, and umami tastes. On the other hand, in a two-bottle test, avoidance of bitter and salty tastes increased, while preference for umami decreased with aging. To investigate the participation of peripheral taste detection systems in the observed changes, we analyzed both the expression of representative taste-related molecules and also turnover rates of taste bud cells. The mRNA expressions of the bitter taste receptor Tas2r105 and its coupled G protein gustducin were significantly decreased with aging. However, the majority of molecules tested did not show significant expression changes. In addition, no significant differences in the turnover rates of taste bud cells were observed between the two age groups. These results suggest that the changes in taste sensitivity of SAMP1 mice due to aging are caused by factors other than the deterioration of taste detection systems in the oral cavity.

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin-proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.

Effect of dietary docosahexaenoic acid on rhodopsin content and packing in photoreceptor cell membranes.

Docosahexaenoic acid (DHA) is enriched in photoreceptor cell membranes. DHA deficiency impairs vision due to photoreceptor cell dysfunction, which is caused, at least in part, by reduced activity of rhodopsin, the light receptor that initiates phototransduction. It is unclear how the depletion of membrane DHA impacts the structural properties of rhodopsin and, in turn, its activity. Atomic force microscopy (AFM) was used to assess the impact of DHA deficiency on membrane structure and rhodopsin organization. AFM revealed that signaling impairment in photoreceptor cells is independent of the oligomeric status of rhodopsin and causes adaptations in photoreceptor cells where the content and density of rhodopsin in the membrane is increased. Functional and structural changes caused by DHA deficiency were reversible.

Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration.

Autophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5Δrod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the α subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5Δrod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and ARR/arrestin are associated with autophagosome-specific proteins. Altogether, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo.

The aglycone of ginsenoside Rg3 enables glucagon-like peptide-1 secretion in enteroendocrine cells and alleviates hyperglycemia in type 2 diabetic mice.

Ginsenosides can be classified on the basis of the skeleton of their aglycones. Here, we hypothesized that the sugar moieties attached to the dammarane backbone enable binding of the ginsenosides to the sweet taste receptor, eliciting glucagon-like peptide-1 (GLP-1) secretion in the enteroendocrine L cells. Using the human enteroendocrine NCI-H716 cells, we demonstrated that 15 ginsenosides stimulate GLP-1 secretion according to the position of their sugar moieties. Through a pharmacological approach and RNA interference technique to inhibit the cellular signal cascade and using the Gαgust(-/-) mice, we elucidated that GLP-1 secreting effect of Rg3 mediated by the sweet taste receptor mediated the signaling pathway. Rg3, a ginsenoside metabolite that transformed the structure through a steaming process, showed the strongest GLP-1 secreting effects in NCI-H716 cells and also showed an anti-hyperglycemic effect on a type 2 diabetic mouse model through increased plasma GLP-1 and plasma insulin levels during an oral glucose tolerance test. Our study reveals a novel mechanism where the sugar moieties of ginsenosides Rg3 stimulates GLP-1 secretion in enteroendocrine L cells through a sweet taste receptor-mediated signal transduction pathway and thus has an anti-hyperglycemic effect on the type 2 diabetic mouse model.

Transcriptional co-factor Transducin beta-like (TBL) 1 acts as a checkpoint in pancreatic cancer malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC.

Photoresponse diversity among the five types of intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate non-image-forming visual responses, including pupillary constriction, circadian photoentrainment and suppression of pineal melatonin secretion. Five morphological types of ipRGCs, M1-M5, have been identified in mice. In order to understand their functions better, we studied the photoresponses of all five cell types, by whole-cell recording from fluorescently labelled ipRGCs visualized using multiphoton microscopy. All ipRGC types generated melanopsin-based ('intrinsic') as well as synaptically driven ('extrinsic') light responses. The intrinsic photoresponses of M1 cells were lower threshold, higher amplitude and faster than those of M2-M5. The peak amplitudes of extrinsic light responses differed among the ipRGC types; however, the responses of all cell types had comparable thresholds, kinetics and waveforms, and all cells received rod input. While all five types exhibited inhibitory amacrine-cell and excitatory bipolar-cell inputs from the 'on' channel, M1 and M3 received additional 'off'-channel inhibition, possibly through their 'off'-sublamina dendrites. The M2-M5 ipRGCs had centre-surround-organized receptive fields, implicating a capacity to detect spatial contrast. In contrast, the receptive fields of M1 cells lacked surround antagonism, which might be caused by the surround of the inhibitory input nullifying the surround of the excitatory input. All ipRGCs responded robustly to a wide range of motion speeds, and M1-M4 cells appeared tuned to different speeds, suggesting that they might analyse the speed of motion. Retrograde labelling revealed that M1-M4 cells project to the superior colliculus, suggesting that the contrast and motion information signalled by these cells could be used by this sensorimotor area to detect novel objects and motion in the visual field.

Expression and subcellular distribution of UNC119a, a protein partner of transducin α subunit in rod photoreceptors.

A recently discovered interaction of rod transducin α subunit (Gα(t1)) with UNC119a is thought to be important for transducin trafficking in photoreceptors. In this study, we analyzed the subcellular distribution of UNC119a under different conditions of illumination in vivo. Analyses by immunofluorescence and Western blotting of retina serial tangential sections demonstrated that UNC119a resides predominantly in the rod inner segment, with a small fraction of UNC119a also appearing to infiltrate the rod outer segment. Such a distribution is consistent with the proposed role of UNC119a in facilitating transducin transport from the rod inner segment to the outer segment in the dark. In addition, UNC119a was present in smaller amounts in the cell body and synaptic region of rods. The profile of UNC119a subcellular distribution remained largely unchanged under all tested conditions of illumination, and correlated with the profile of Gα(t1) following its light-dependent translocation. Quantification by Western blotting suggested that mouse retina contains ~17 pmol of UNC119a, giving a ~1 to 4 molar ratio of UNC119a to Gα(t1). Hence, light-translocated Gα(t1) can serve as a major partner of UNC119a. Supporting this role, the levels of UNC119a were downregulated by about 2-fold in mouse retina lacking Gα(t1). As a dominant partner, Gα(t1) may potentially modulate the function of other known UNC119a-interacting proteins involved in photoreceptor ciliary trafficking and synaptic regulation, in a light-dependent manner.

Restoration of vision after transplantation of photoreceptors.

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Gα-gustducin is extensively coexpressed with sweet and bitter taste receptors in both the soft palate and fungiform papillae but has a different functional significance.

To clarify the regional differences in the expression and functional significance of Gα-gustducin in soft palate (SP) and fungiform (FF) taste buds, we examined the coexpression of Gα-gustducin with taste receptors and the impact of Gα-gustducin knockout (gKO) on neural responses to several sweet and bitter compounds. Sweet responses from both the greater superficial petrosal (GSP) and chorda tympani (CT) nerves in gKO mice were markedly depleted, reflecting overlapping expression of Gα-gustducin and Tas1r2. However, although Gα-gustducin was expressed in 87% and 88% of Tas2rs cells in the SP and FF, respectively, there were no statistically significant differences in the CT responses to quinine-HCl (QHCl) and denatonium (Den) between gKO and wild-type (WT) mice. In contrast, GSP responses to these compounds were markedly reduced in gKO mice with an apparent elevation of thresholds (>10-fold). These results suggest that 1) Gα-gustducin plays a critical role in sweet transduction in both the SP and the FF, 2) other Gα subunits coexpressed with Gα-gustducin in the FF are sufficient for responses to QHCl and Den, and 3) robust GSP responses to QHCl and Den occur in the SP by a Gα-gustducin-dependent mechanism, which is absent in the FF.

UNC119 is required for G protein trafficking in sensory neurons.

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like ß-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities.

In mammals, synchronization of the circadian pacemaker in the hypothalamus is achieved through direct input from the eyes conveyed by intrinsically photosensitive retinal ganglion cells (ipRGCs). Circadian photoentrainment can be maintained by rod and cone photoreceptors, but their functional contributions and their retinal circuits that impinge on ipRGCs are not well understood. Using mice that lack functional rods or in which rods are the only functional photoreceptors, we found that rods were solely responsible for photoentrainment at scotopic light intensities. Rods were also capable of driving circadian photoentrainment at photopic intensities at which they were incapable of supporting a visually guided behavior. Using mice in which cone photoreceptors were ablated, we found that rods signal through cones at high light intensities, but not at low light intensities. Thus, rods use two distinct retinal circuits to drive ipRGC function to support circadian photoentrainment across a wide range of light intensities.

The OFF-pathway dominance of P2X(2)-purinoceptors is formed without visual experience.

Visual input in the critical period is an important determinant of the functions of the visual system, affecting for example the formation of the ocular dominance column in the visual cortex. The final map of columnar organization is usually determined by plastic changes in the critical period, but organization is distorted without adequate visual input. Here, we examined whether formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling in the mouse retina is the result of visual experience. The P2X(2)-purinoceptor signaling pathway developed during the critical period. However, visual experience in this period produced no plastic change in the formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling. Our findings suggest that the OFF-pathway dominance of P2X(2)-signaling in the mouse retina is intrinsically programmed.

Influence of the rod photoresponse on light adaptation and circadian rhythmicity in the cone ERG.

PURPOSE: In the mammalian retina, rod and cone pathways are fundamentally intertwined, with signals from both converging on cone bipolar cells to reach retinal ganglion cells. Psychophysical and electrophysiological data suggests that, as a consequence, rod signal transduction has a suppressive effect on the activity of cone pathways. It therefore might be assumed that the balance between rod and cone input to cone bipolar cells would be subject to dynamic regulation. There is evidence of light and time-of-day dependent alterations in this parameter. Here we set out to determine the extent to which such changes in rod-cone pathway convergence explain alterations in cone pathway function associated with light adaptation and circadian phase by recording cone electroretinograms (ERGs) in mice deficient in rod phototransduction. METHODS: Cone-isolated ERGs elicited by bright flashes superimposed on a rod saturating background light were recorded from wild-type and rod transducin deficient (Gnat1(-/-)) mice. The process of light adaptation was observed by tracing changes in the ERG waveform over 20 min exposure to the background light in these genotypes, and circadian control by comparing responses at subjective midday and midnight. RESULTS: The cone ERG b-wave exhibited significantly enhanced amplitude and reduced latency (implicit time) in Gnat1(-/-) mice under all conditions. Light adaptation was associated with a robust increase in b-wave amplitude in Gnat1(-/-) mice but, in contrast to wild types, almost no change in implicit time. Gnat1(-/-) mice retained circadian rhythms in the cone ERG with b-wave amplitudes larger and latencies reduced during the subjective day. CONCLUSIONS: Rod phototransduction has a strong suppressive effect on the cone ERG. Light adaptation in cone pathways relies in part on reductions in this effect, although mechanisms intrinsic to cone pathways also play an important role. Similarly, while changes in coupling between rod and cone pathways over the course of the day may contribute to circadian regulation of the cone pathway they are not sufficient to explain circadian rhythms in the wild-type cone ERG.

Transducin gamma-subunit sets expression levels of alpha- and beta-subunits and is crucial for rod viability.

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the alpha- and betagamma-subunits. Here we demonstrate that the knock-out of transducin gamma-subunit leads to a major downregulation of both alpha- and beta-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining alpha- and beta-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the alpha-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the gamma-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin beta-subunit without its constitutive gamma-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

N-terminal fatty acylation of transducin profoundly influences its localization and the kinetics of photoresponse in rods.

N-terminal acylation of the alpha-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein alpha-subunit, transducin (G alpha(t)), we generated transgenic mice that expressed a mutant G alpha(t) lacking N-terminal acylation sequence (G alpha(t)G2A). Rods expressing G alpha(t)G2A showed a severe defect in transducin cellular localization. In contrast to native G alpha(t), which resides in the outer segments of dark-adapted rods, G alpha(t)G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G alpha(t)G2A at 5-6% of the G alpha(t) levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G alpha(t)G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G alpha(t)G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G alpha(t) membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

Residual cone vision without alpha-transducin.

Behavioral experiments in humans with a rare genetic mutation that compromises the function of alpha-transducin (Galpha the alpha-subunit of the G-protein in the primary cone phototransduction cascade) reveal a residual cone response only viable at high light levels and at low temporal frequencies. It has three characteristic properties. First, it limits temporal frequency sensitivity to the equivalent of a simple first order reaction with a time constant of approximately 140 ms. Second, it delays the visual response by an amount that is also consistent with such a reaction. Third, it causes temporal acuity to be linearly related to the logarithm of the amount of bleached pigment. We suggest that these properties are consistent with the residual function depending on a sluggishly generated cone photobleaching product, which we tentatively identify as a cone metarhodopsin. By activating the transduction cascade, this bleaching product mimics the effects of real light and is therefore one of the molecular origins of "background equivalence," the long-established observation that the aftereffects of photopigment bleaches and the effects of real background lights are equivalent. Alternative explanations for the residual cone response include the possibilities that there is a secondary phototransduction mechanism that bypasses alpha-transduction, or that the truncated alpha-transduction that results from the mutation retains some minimal functionality.

Taste responses to sweet stimuli in alpha-gustducin knockout and wild-type mice.

The importance of alpha-gustducin in sweet taste transduction is based on data obtained with sucrose and the artificial sweetener SC45647. Here we studied the role of alpha-gustducin in sweet taste. We compared the behavioral and electrophysiological responses of alpha-gustducin knockout (KO) and wild-type (WT) mice to 11 different sweeteners, representing carbohydrates, artificial sweeteners, and sweet amino acids. In behavioral experiments, over 48-h preference ratios were measured in two-bottle preference tests. In electrophysiological experiments, integrated responses of chorda tympani (CT) and glossopharyngeal (NG) nerves were recorded. We found that preference ratios of the KO mice were significantly lower than those of WT for acesulfame-K, dulcin, fructose, NC00174, D-phenylalanine, L-proline, D-tryptophan, saccharin, SC45647, sucrose, but not neotame. The nerve responses to all sweeteners, except neotame, were smaller in the KO mice than in the WT mice. The differences between the responses in WT and KO mice were more pronounced in the CT than in the NG. These data indicate that alpha-gustducin participates in the transduction of the sweet taste in general.