Differential impact of Kv8.2 loss on rod and cone signaling and degeneration.

Heteromeric Kv2.1/Kv8.2 channels are voltage-gated potassium channels localized to the photoreceptor inner segment. They carry IKx, which is largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset cone dystrophy with supernormal rod response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms, we show that there were delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels were controlled by the perfusion buffer, so we propose the in vivo alteration is influenced by genotype-associated ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone-driven ERG b wave amplitudes and underwent degeneration. Altogether, we provide insight into how a deficit in the dark current affects the health and function of photoreceptors.

Cone-Driven Retinal Responses Are Shaped by Rod But Not Cone HCN1.

Signal integration of converging neural circuits is poorly understood. One example is in the retina where the integration of rod and cone signaling is responsible for the large dynamic range of vision. The relative contribution of rods versus cones is dictated by a complex function involving background light intensity and stimulus temporal frequency. One understudied mechanism involved in coordinating rod and cone signaling onto the shared retinal circuit is the hyperpolarization activated current (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels expressed in rods and cones. Ih opposes membrane hyperpolarization driven by activation of the phototransduction cascade and modulates the strength and kinetics of the photoreceptor voltage response. We examined conditional knock-out (KO) of HCN1 from mouse rods using electroretinography (ERG). In the absence of HCN1, rod responses are prolonged in dim light which altered the response to slow modulation of light intensity both at the level of retinal signaling and behavior. Under brighter intensities, cone-driven signaling was suppressed. To our surprise, conditional KO of HCN1 from mouse cones had no effect on cone-mediated signaling. We propose that Ih is dispensable in cones because of the high level of temporal control of cone phototransduction. Thus, HCN1 is required for cone-driven retinal signaling only indirectly by modulating the voltage response of rods to limit their output.SIGNIFICANCE STATEMENT Hyperpolarization gated hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry a feedback current that helps to reset light-activated photoreceptors. Using conditional HCN1 knock-out (KO) mice we show that ablating HCN1 from rods allows rods to signal in bright light when they are normally shut down. Instead of enhancing vision this results in suppressing cone signaling. Conversely, ablating HCN1 from cones was of no consequence. This work provides novel insights into the integration of rod and cone signaling in the retina and challenges our assumptions about the role of HCN1 in cones.

Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.

α2δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses.

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

Analysis of 14-3-3 isoforms expressed in photoreceptors.

The 14-3-3 family of proteins has undergone considerable expansion in higher eukaryotes with humans and mice expressing seven isoforms (ß, ε, η, γ, θ, ζ, and σ) from seven distinct genes (YWHAB, YWAHE, YWHAH, YWHAG, YWHAQ, YWHAZ, and SFN). Growing evidence indicates that while highly conserved, these isoforms are not entirely functionally redundant as they exhibit unique tissue expression profiles, subcellular localization, and biochemical functions. A key limitation in our understanding of 14-3-3 biology lies in our limited knowledge of cell-type specific 14-3-3 expression. Here we provide a characterization of 14-3-3 expression in whole retina and isolated rod photoreceptors using reverse-transcriptase digital droplet PCR. We find that all 14-3-3 genes with the exception of SFN are expressed in mouse retina with YWHAQ and YWHAE being the most highly expressed. Rod photoreceptors are enriched in YWHAE (14-3-3 ε). Immunohistochemistry revealed that 14-3-3 ε and 14-3-3 ζ exhibit unique distributions in photoreceptors with 14-3-3 ε restricted to the inner segment and 14-3-3 ζ localized to the outer segment. Our data demonstrates that, in the retina, 14-3-3 isoforms likely serve specific functions as they exhibit unique expression levels and cell-type specificity. As such, future investigations into 14-3-3 function in rod photoreceptors should be centered on 14-3-3 ε and 14-3-3 ζ, depending on the subcellular region of question.

Identification of a VxP Targeting Signal in the Flagellar Na+ /K+ -ATPase.

Na(+) /K(+) -ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes-specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin-B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co-immunoprecipitates with ankyrin-B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP-NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes-specific NKA isoform.

A di-arginine ER retention signal regulates trafficking of HCN1 channels from the early secretory pathway to the plasma membrane.

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry Ih, which contributes to neuronal excitability and signal transmission in the nervous system. Controlling the trafficking of HCN1 is an important aspect of its regulation, yet the details of this process are poorly understood. Here, we investigated how the C-terminus of HCN1 regulates trafficking by testing for its ability to redirect the localization of a non-targeted reporter in transgenic Xenopus laevis photoreceptors. We found that HCN1 contains an ER localization signal and through a series of deletion constructs, identified the responsible di-arginine ER retention signal. This signal is located in the intrinsically disordered region of the C-terminus of HCN1. To test the function of the ER retention signal in intact channels, we expressed wild type and mutant HCN1 in HEK293 cells and found this signal negatively regulates surface expression of HCN1. In summary, we report a new mode of regulating HCN1 trafficking: through the use of a di-arginine ER retention signal that monitors processing of the channel in the early secretory pathway.

Clinical and cytokine responses to house dust mite sublingual immunotherapy.

BACKGROUND: Cytokine responses accompanying sublingual immunotherapy (SLIT) responder phenotypes have not previously been reported. OBJECTIVE: To investigate clinical and cytokine responses of house dust mite (HDM) sensitive patients with allergic rhinitis receiving HDM SLIT or placebo for 2 years. METHODS: Sixty adults were randomized to receive SLIT or placebo. Clinical symptoms were measured using the Total 5 Symptom Score (TSS5) and Juniper Rhinitis Quality of Life Questionnaire. HDM specific IgE, IgG, skin prick tests, and HDM-stimulated release of interleukin (IL) 5 and interferon γ (IFN-γ) in peripheral blood mononuclear cells was studied at 0, 6, 12, and 24 months and IL-13, IL-4, and IL-10 at 0 and 24 months. RESULTS: A total of 32 of 39 SLIT and 16 of 21 placebo patients completed the study. There was significant clinical improvement in both the SLIT and placebo groups. Median T5SS decreased from 14.75 to 5.25 in the SLIT group (P < .001) and 12.7 to 6.0 in the placebo group (P = .003). The median quality-of-life score also decreased in the SLIT group (P < .001) and the placebo group (P < .001). A subgroup analysis of patients found a 60% or greater improvement (on the T5SS and the Juniper Rhinitis Quality of Life Questionnaire) in the good responders group and a 30% to 59% improvement or no improvement in the intermediate responders group. This subgroup analysis also found more good responders in the SLIT group (47%) compared with the placebo group (25%; P = .07). Significant decreases in the IL-5/IFN-γ (P < .001), IL-13/IFN-γ (P < .001), and IL-4/IFN-γ (P = .03) ratios were found in the combined good clinical improvement group at 24 months. CONCLUSION: A good clinical response (≥60% improvement in both TSS5 and quality of life) is associated with significant decreases in IL-5, IL-13, and IL-4 relative to IFN-γ during 2 years of SLIT therapy for HDMs.

Ranking solvent interactions and dielectric constants with [Pt(mesBIAN)(tda)]: A cautionary tale for polarity determinations in ionic liquids.

The solvatochromic properties of [Pt(mesBIAN)(tda)] are studied in traditional molecular solvents and ionic liquids and duly compared along established empirical solvent parameter scales. The charge-transfer absorption band of [Pt(mesBIAN)(tda)] is determined to be primarily dependent upon solvent acidity and dipolarity. Notably, ionic liquids do not obey the same well-behaved trend as molecular solvents, highlighting the complexity and domain (nano)segregation inherent to ionic liquids.

Photoreceptor inner and outer segments.

Photoreceptors are exquisitely adapted to transform light stimuli into electrical signals that modulate neurotransmitter release. These cells are organized into several compartments including the unique outer segment (OS). Its whole function is to absorb light and transduce this signal into a change of membrane potential. Another compartment is the inner segment where much of metabolism and regulation of membrane potential takes place and that connects the OS and synapse. The synapse is the compartment where changes in membrane potentials are relayed to other neurons in the retina via release of neurotransmitter. The composition of the plasma membrane surrounding these compartments varies to accommodate their specific functions. In this chapter, we discuss the organization of the plasma membrane emphasizing the protein composition of each region as it relates to visual signaling. We also point out examples where mutations in these proteins cause visual impairment.

Mouse all-cone retina models of Cav1.4 synaptopathy.

The voltage-gated calcium channel, Cav1.4 is localized to photoreceptor ribbon synapses and functions both in molecular organization of the synapse and in regulating release of synaptic vesicles. Mutations in Cav1.4 subunits typically present as either incomplete congenital stationary night blindness or a progressive cone-rod dystrophy in humans. We developed a cone-rich mammalian model system to further study how different Cav1.4 mutations affect cones. RPE65 R91W KI; Nrl KO "Conefull" mice were crossed to Cav1.4 α1F or α2δ4 KO mice to generate the "Conefull:α1F KO" and "Conefull:α2δ4 KO" lines. Animals were assessed using a visually guided water maze, electroretinogram (ERG), optical coherence tomography (OCT), and histology. Mice of both sexes and up to six-months of age were used. Conefull: α1F KO mice could not navigate the visually guided water maze, had no b-wave in the ERG, and the developing all-cone outer nuclear layer reorganized into rosettes at the time of eye opening with degeneration progressing to 30% loss by 2-months of age. In comparison, the Conefull: α2δ4 KO mice successfully navigated the visually guided water maze, had a reduced amplitude b-wave ERG, and the development of the all-cone outer nuclear layer appeared normal although progressive degeneration with 10% loss by 2-months of age was observed. In summary, new disease models for studying congenital synaptic diseases due to loss of Cav1.4 function have been created.

A visually guided swim assay for mouse models of human retinal disease recapitulates the multi-luminance mobility test in humans.

PURPOSE: The purpose of this study was to develop a visually guided swim assay (VGSA) for measuring vision in mouse retinal disease models comparable to the multi-luminance mobility test (MLMT) utilized in human clinical trials. METHODS: Three mouse retinal disease models were studied: Bardet-Biedl syndrome type 1 (Bbs1M390R/M390R), n = 5; Bardet-Biedl syndrome type 10 (Bbs10-/-), n = 11; and X linked retinoschisis (retinoschisin knockout; Rs1-KO), n = 5. Controls were normally-sighted mice, n = 10. Eyeless Pax6Sey-Dey mice, n = 4, were used to determine the performance of animals without vision in VGSA. RESULTS: Eyeless Pax6Sey-Dey mice had a VGSA time-to-platform (TTP) 7X longer than normally-sighted controls (P < 0.0001). Controls demonstrated no difference in their TTP in both lighting conditions; the same was true for Pax6Sey-Dey. At 4-6 M, Rs1-KO and Bbs10-/- had longer TTP in the dark than controls (P = 0.0156 and P = 1.23 × 10-8, respectively). At 9-11 M, both BBS models had longer TTP than controls in light and dark with times similar to Pax6Sey-Dey (P < 0.0001), demonstrating progressive vision loss in BBS models, but not in controls nor in Rs1-KO. At 1 M, Bbs10-/- ERG light-adapted (cone) amplitudes were nonrecordable, resulting in a floor effect. VGSA did not reach a floor until 9-11 M. ERG combined rod/cone b-wave amplitudes were nonrecordable in all three mutant groups at 9-11 M, but VGSA still showed differences in visual function. ERG values correlate non-linearly with VGSA, and VGSA measured the continual decline of vision. CONCLUSION: ERG is no longer a useful endpoint once the nonrecordable level is reached. VGSA differentiates between different levels of vision, different ages, and different disease models even after ERG is nonrecordable, similar to the MLMT in humans.

Membrane attachment is key to protecting transducin GTPase-activating complex from intracellular proteolysis in photoreceptors.

The members of the R7 regulator of G-protein signaling (RGS) protein subfamily are versatile regulators of G-protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins that serve as versatile modulators of their activity, intracellular targeting, and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1 · Gß5 GTPase-activating complex responsible for the rapid inactivation of the G-protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gß5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1 · Gß5 to cellular membranes. Furthermore, membrane attachment of RGS9-1 · Gß5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1 · Gß5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP · RGS9-1 · Gß5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.

Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.

Fluorescent probe studies of polarity and solvation within room temperature ionic liquids: a review.

Ionic liquids display an array of useful and sometimes unconventional, solvent features and have attracted considerable interest in the field of green chemistry for the potential they hold to significantly reduce environmental emissions. Some of these points have a bearing on the chemical reactivity of these systems and have also generated interest in the physical and theoretical aspects of solvation in ionic liquids. This review presents an introduction to the field of ionic liquids, followed by discussion of investigations into the solvation properties of neat ionic liquids or mixed systems including ionic liquids as a major or minor component. The ionic liquid based multicomponent systems discussed are composed of other solvents, other ionic liquids, carbon dioxide, surfactants or surfactant solutions. Although we clearly focus on fluorescence spectroscopy as a tool to illuminate ionic liquid systems, the issues discussed herein are of general relevance to discussions of polarity and solvent effects in ionic liquids. Transient solvation measurements carried out by means of time-resolved fluorescence measurements are particularly powerful for their ability to parameterize the kinetics of the solvation process in ionic liquids and are discussed as well.

Binding of the ionic liquid cation 1-alkyl-3-methylimidazolium to p-tetranitrocalix[4]arene probed by fluorescent indicator displacement.

Acridine orange (AO) was used as a fluorescent probe molecule to study the encapsulation of an alkylimidazolium cation from a water-soluble ionic liquid (IL) within two cavitand species, p-tetranitrocalix[4]arene (1) and calix[4]resorcinarene (2), both in alkaline aqueous media. The addition of IL to the preformed [1·AO] adduct resulted in significantly increased fluorescence due to the expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of the 1-alkyl-3-methylimidazolium cation ([C(n)mim](+), n = 4 and 6) by 1. Conversely, the fluorescence signal dropped upon the addition of IL to the [2·AO] host-guest complex due to unfavorable binding between [C(n)mim](+) and 2. The formation of these postulated adducts is corroborated using ab initio calculations, which also provide evidence for the location of [bmim](+) at the lower external rim of [2·AO], providing an explanation for the observed luminescence quenching in the latter case. These results point to a number of different paths for exploration, ranging from the fluorescence monitoring of IL contamination in groundwater to the "daisy chaining" of macrocyles toward supramolecular ionic networks. They also broadly encourage the exploration of ILs in host-guest-based optical and mass spectrometric sensory systems.

Computational prediction of ionic liquid 1-octanol/water partition coefficients.

Wet 1-octanol/water partition coefficients (log K(ow)) predicted for imidazolium-based ionic liquids using adaptive bias force-molecular dynamics (ABF-MD) simulations lie in excellent agreement with experimental values. These encouraging results suggest prospects for this computational tool in the a priori prediction of log K(ow) values of ionic liquids broadly with possible screening implications as well (e.g., prediction of CO(2)-philic ionic liquids).

Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC-MALS-SAXS and negative-stain electron microscopy. The Kv2.1 T1-T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Identification of HCN1 as a 14-3-3 client.

Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed, as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding peptides is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of mouse S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

OBJECTIVE: To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. METHODS: AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1µL) or subretinal (N = 21; 2µL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. RESULTS: The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. CONCLUSIONS: AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.