Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration.

Purpose: Electric micro-current has been shown to enhance penetration and transduction of adeno-associated viral (AAV) vectors in mouse retina after intravitreal administration. We termed this: "electric-current vector mobility (ECVM)." The present study considered whether ECVM could augment retinal transduction efficiency of intravitreal AAV8-CMV-EGFP in normal rabbit and nonhuman primate (NHP) macaque. Potential mechanisms underlying enhanced retinal transduction by ECVM were also studied. Methods: We applied an electric micro-current across the intact eye of normal rabbit and monkey in vivo for a brief period immediately after intravitreal injection of AAV8-CMV-EGFP. Retinal GFP expression was evaluated by fundus imaging in vivo. Retinal immunohistochemistry was performed to assess the distribution of retinal cells transduced by the AAV8-EGFP. Basic fibroblast growth factor (bFGF) was analyzed by quantitative RT-polymerase chain reaction (PCR). Müller glial reactivity and inner limiting membrane (ILM) were examined by the glial fibrillary acidic protein (GFAP) and vimentin staining in mouse retina, respectively. Results: ECVM significantly increased the efficiency of AAV reaching and transducing the rabbit retina following intravitreal injection, with gene expression in inner nuclear layer, ganglion cells, and Müller cells. Similar trend of improvement was observed in the ECVM-treated monkey eye. The electric micro-current upregulated bFGF expression in Müller cells and vimentin showed ILM structural changes in mouse retina. Conclusions: ECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection. Translational Relevance: This work has potential translational relevance to human ocular gene therapy by increasing retinal expression of therapeutic vectors given by intravitreal administration.

Trans-ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Gene Transduction after Intravitreal Vector Administration.

Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina. Unfortunately, most conventional, non-modified AAV vector serotypes penetrate the retina poorly from the vitreous; this limits efficient transduction and expression by target cells (retinal pigment epithelium and photoreceptors). We developed a method of applying a small and safe electric current across the intact eye in vivo for a brief period following intravitreal vector administration. This significantly improved AAV-mediated transduction of retinal cells in wild-type mice following intravitreal delivery, with gene expression in retinal pigment epithelium and photoreceptor cells. The low-level current had no adverse effects on retinal structure and function. This method should be generally applicable for other AAV serotypes and may have broad application in both basic research and clinical studies.

Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography.

PURPOSE: Spectral-domain optical coherence tomography (SD-OCT) was used to characterize the retinal phenotype, natural history, and treatment responses in a mouse model of X-linked retinoschisis (Rs1-KO) and to identify new structural markers of AAV8-mediated gene therapy outcome. METHODS: Optical coherence tomography scans were performed on wild-type and Rs1-KO mouse retinas between 1 and 12 months of age and on Rs1-KO mice after intravitreal injection of AAV8-scRS/IRBPhRS (AAV8-RS1). Cavities and photoreceptor outer nuclear layer (ONL) thickness were measured, and outer retina reflective band (ORRB) morphology was examined with age and after AAV8-RS1 treatment. Outer retina reflective band morphology was compared to immunohistochemical staining of the outer limiting membrane (OLM) and photoreceptor inner segment (IS) mitochondria and to electron microscopy (EM) images of IS. RESULTS: Retinal cavity size in Rs1-KO mice increased between 1 and 4 months and decreased thereafter, while ONL thickness declined steadily, comparable to previous histologic studies. Wild-type retina had four ORRBs. In Rs1-KO, ORRB1was fragmented from 1 month, but was normal after 8 months; ORRB2 and ORRB3 were merged at all ages. Outer retina reflective band morphology returned to normal after AAV-RS1 therapy, paralleling the recovery of the OLM and IS mitochondria as indicated by anti-ß-catenin and anti-COX4 labeling, respectively, and EM. CONCLUSIONS: Spectral-domain OCT is a sensitive, noninvasive tool to monitor subtle changes in retinal morphology, disease progression, and effects of therapies in mouse models. The ORRBs may be useful to assess the outcome of gene therapy in the treatment of X-linked retinoschisis patients.

NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice.

PURPOSE: The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1. METHODS: Transgenic (Tg)-CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. RESULTS: Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP-transduced retinas. CONCLUSIONS: Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway.

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.

Transgenic expression of constitutively active RAC1 disrupts mouse rod morphogenesis.

PURPOSE: Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1. METHODS: Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy. Function was evaluated by electroretinography. Potential protein partners of CA RAC1 were identified by co-immunoprecipitation of retinal extracts. RESULTS: Constitutively active RAC1 expression in differentiating rods disrupted outer retinal lamination as early as postnatal day (P)6, and many photoreceptor cell nuclei were displaced apically into the presumptive subretinal space. These photoreceptors did not develop normal inner and outer segments and had abnormal placement of synaptic elements. Some photoreceptor nuclei were also mislocalized into the inner nuclear layer. Extensive photoreceptor degeneration was subsequently observed in the adult animal. Constitutively active RAC1 formed a complex with the polarity protein PAR6 and with microtubule motor dynein in mouse retina. The normal localization of the PAR6 complex was disrupted in CA RAC1-expressing rod photoreceptors. CONCLUSIONS: Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. PAR6 and dynein are two potential downstream effectors that may be involved in CA RAC1-mediated defective mouse photoreceptor morphogenesis.

Phosphorylation of phosducin accelerates rod recovery from transducin translocation.

PURPOSE: In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin ßγ subunits in its de-phosphorylated state, in regulating this process. METHODS: Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina. RESULTS: In mice expressing normal phosducin, transducin α and ßγ subunits returned to the outer segments with a half-time (t(1/2)) of ∼24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin α subunit moved four times slower, with t(1/2) ∼95 minutes, while the movement of transducin ßγ was less affected. CONCLUSIONS: We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin α subunit in the outer segment is driven by its re-binding to the transducin ßγ dimer, because this process is accelerated significantly by phosducin phosphorylation.

Current understanding of usher syndrome type II.

Usher syndrome is the most common deafness-blindness caused by genetic mutations. To date, three genes have been identified underlying the most prevalent form of Usher syndrome, the type II form (USH2). The proteins encoded by these genes are demonstrated to form a complex in vivo. This complex is localized mainly at the periciliary membrane complex in photoreceptors and the ankle-link of the stereocilia in hair cells. Many proteins have been found to interact with USH2 proteins in vitro, suggesting that they are potential additional components of this USH2 complex and that the genes encoding these proteins may be the candidate USH2 genes. However, further investigations are critical to establish their existence in the USH2 complex in vivo. Based on the predicted functional domains in USH2 proteins, their cellular localizations in photoreceptors and hair cells, the observed phenotypes in USH2 mutant mice, and the known knowledge about diseases similar to USH2, putative biological functions of the USH2 complex have been proposed. Finally, therapeutic approaches for this group of diseases are now being actively explored.

Disruption of the chaperonin containing TCP-1 function affects protein networks essential for rod outer segment morphogenesis and survival.

Type II Chaperonin Containing TCP-1 (CCT, also known as TCP-1 Ring Complex, TRiC) is a multi-subunit molecular machine thought to assist in the folding of ∼ 10% of newly translated cytosolic proteins in eukaryotes. A number of proteins folded by CCT have been identified in yeast and cultured mammalian cells, however, the function of this chaperonin in vivo has never been addressed. Here we demonstrate that suppressing the CCT activity in mouse photoreceptors by transgenic expression of a dominant-negative mutant of the CCT cofactor, phosducin-like protein (PhLP), results in the malformation of the outer segment, a cellular compartment responsible for light detection, and triggers rapid retinal degeneration. Investigation of the underlying causes by quantitative proteomics identified distinct protein networks, encompassing ∼ 200 proteins, which were significantly affected by the chaperonin deficiency. Notably among those were several essential proteins crucially engaged in structural support and visual signaling of the outer segment such as peripherin 2, Rom1, rhodopsin, transducin, and PDE6. These data for the first time demonstrate that normal CCT function is ultimately required for the morphogenesis and survival of sensory neurons of the retina, and suggest the chaperonin CCT deficiency as a potential, yet unexplored, cause of neurodegenerative diseases.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Serine/threonine kinase akt activation regulates the activity of retinal serine/threonine phosphatases, PHLPP and PHLPPL.

In our previous studies, we have shown that insulin receptor (IR) activation leads to the activation of phosphoinositide 3-kinase (PI3K) and Akt activation in rod photoreceptors. This pathway is functionally important for photoreceptor survival as deletion of IR and one of the isoforms of Akt (Akt2) resulted in stress-induced photoreceptor degeneration. However, the molecular mechanism of this degeneration is not known. Akt signaling is known to be regulated by the serine/threonine phosphatases, PH domain and leucine-rich repeat protein phosphatases (PHLPP) and PHLPP-like (PHLPPL). In this study, we characterized these two phosphatases in the retina and examined the role of IR, PI3K, and Akt signaling on the activity of PHLPP and PHLPPL. Most of the studies published on PHLPP and PHLPPL are directed toward Akt dephosphorylation; however, there are no studies available to date on how the enzyme activities of these phosphatases are regulated. We made a novel finding in this study that both PHLPP and PHLPPL activities were significantly decreased in the presence of insulin ex vivo. The insulin-induced decrease of phosphatase activities were PI3K-dependent as pre-treatment of ex vivo retinal cultures with LY294002 significantly reversed the insulin-induced inhibition. It has been shown previously that PHLPP and PHLPPL regulate the dephosphorylation of Akt isoforms, and our results demonstrate for the first time that retinal PHLPP and PHLPPL activities are under the control of the IR-activated PI3K/Akt pathway.

Analysis of protein expression and compartmentalization in retinal neurons using serial tangential sectioning of the retina.

The progress in understanding visual signal transduction in vertebrate photoreceptors, arguably the best studied G protein-mediated signal transduction cascade in modern biology, was facilitated by the unique anatomy of rod photoreceptors. Held only by thin connected cilia, rod outer segments can be readily separated from the rest of the retina simply by shaking, and then purified by gradient centrifugation. The availability of such an efficient procedure of rod outer segment purification not only previously facilitated the identification of many principal visual signaling proteins located in this cellular compartment, but it is also currently being exploited in proteomics studies. In this paper, we describe a simple and inexpensive technique that allows for the quantitative analysis of protein expression within different subcellular compartments of photoreceptors, and could also be used for studying protein expression in the secondary retinal neurons. This technique is based on the Western blot analysis of the protein content of serial sections obtained by tangential sectioning of flat-mounted frozen retinas from mouse and rat, and it could serve as a way to validate proteomic data, similar to the way the quantitative RT-PCR technique is used for validation of gene-microarray data. To demonstrate the utility of this technique, we have determined the expression profiles in normal mouse retina of several signaling, energy-producing, and chaperone proteins, which were recently identified in bovine rod photoreceptors by mass spectrometry.

Targeting of RGS7/Gbeta5 to the dendritic tips of ON-bipolar cells is independent of its association with membrane anchor R7BP.

Complexes of regulator of G-protein signaling (RGS) proteins with G-protein beta5 (Gbeta5) subunits are essential components of signaling pathways that regulate the temporal characteristics of light-evoked responses in vertebrate retinal photoreceptors and ON-bipolar cells. Recent studies have found that RGS/Gbeta5 complexes bind to a new family of adapter proteins, R9AP (RGS9 anchor protein) and R7 family binding protein (R7BP), that in case of the RGS9/Gbeta5 complex were shown to determine its precise subcellular targeting to either the outer segment of photoreceptors or postsynaptic structures of striatal neurons, respectively. In this study, we establish that another trimeric complex consisting of RGS7, Gbeta5, and R7BP subunits is specifically targeted to the dendritic tips of retinal bipolar cells. However, examination of the mechanisms of complex targeting in vivo surprisingly revealed that the delivery of RGS7/Gbeta5 to the dendrites of ON-bipolar cells occurs independently of its association with R7BP. These findings provide a new mechanism for adapter-independent targeting of RGS/Gbeta5 complexes.

Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation.

For over a decade, phosducin's interaction with the betagamma subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd- / -) rods. Pd- / - rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd- / - rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin betagamma-subunit expression, and thus available heterotrimer in Pd- / - rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.

Compartment-specific phosphorylation of phosducin in rods underlies adaptation to various levels of illumination.

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gbetagamma subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71.

Localization and differential interaction of R7 RGS proteins with their membrane anchors R7BP and R9AP in neurons of vertebrate retina.

G protein signaling in the retina is crucially regulated by the R7 family of regulators of G protein signaling (RGS) proteins, which act to stimulate the rate of G protein inactivation. Recent findings indicate that R7 RGS proteins form complexes with two newly identified membrane anchors: RGS9 Anchor Protein (R9AP) and R7 Binding Protein (R7BP), which play essential roles in modulating the expression and localization of R7 RGS proteins. Here we demonstrate that the four R7 RGS proteins: RGS6, RGS7, RGS9 and RGS11 differentially associate with two membrane anchors. R9AP was found to form complexes with RGS9 and RGS11 which were substantially enriched in the photoreceptors. In contrast, complexes of R7BP with R7 RGS proteins were predominantly localized to the synaptic projections of retina neurons, suggesting their involvement in regulation of synaptic transmission between retina neurons. Furthermore, studies of knockout mice revealed that R9AP is necessary for the expression of only RGS9 but not for RGS6, 7 or 11. Together these data suggest that R7 RGS proteins in the retina are present as macromolecular complexes with their membrane anchors that could differentially regulate their function in various retina neurons.

Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin alpha subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

The tetraspanin protein peripherin-2 forms a complex with melanoregulin, a putative membrane fusion regulator.

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.