Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3-13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.

Dogs and humans share a common susceptibility gene SRBD1 for glaucoma risk.

Glaucoma is a degenerative optic neuropathy that is associated with elevated intraocular pressure. Primary open angle glaucoma is the most common type of glaucoma in canines, and its highest incidence among dog breeds has been reported in Shiba-Inus, followed by Shih-Tzus. These breeds are known to have an abnormal iridocorneal angle and dysplastic prectinate ligament. However, the hereditary and genetic backgrounds of these dogs have not yet been clarified. In this study, we investigated the association between polymorphisms of the glaucoma candidate genes, SRBD1, ELOVL5, and ADAMTS10, and glaucoma in Shiba-Inus and Shih-Tzus. We analyzed 11 polymorphisms in these three genes using direct DNA sequencing. Three SRBD1 SNPs, rs8655283, rs22018514 and rs22018513 were significantly associated with glaucoma in Shiba-Inus, while rs22018513, a synonymous SNP in exon 4, showed the strongest association (P = 0.00039, OR = 3.03). Conditional analysis revealed that rs22018513 could account for most of the association of these SNPs with glaucoma in Shiba-Inus. In Shih-Tzus, only rs9172407 in the SRBD1 intron 1 was significantly associated with glaucoma (P = 0.0014, OR = 5.25). There were no significant associations between the ELOVL5 or ADAMTS10 polymorphisms and glaucoma in Shiba-Inus and Shih-Tzus. The results showed that SRBD1 polymorphisms play an important role in glaucoma pathology in both Shiba-Inus and Shih-Tzus. SRBD1 polymorphisms have also been associated with normal- and high-tension glaucomas in humans. Therefore, SRBD1 may be a common susceptibility gene for glaucoma in humans and dogs. We anticipate that the nucleotide sequencing data from this study can be used in genetic testing to determine for the first time, the genetic status and susceptibility of glaucoma in dogs, with high precision. Moreover, canine glaucoma resulting from SRBD1 polymorphisms could be a useful animal model to study human glaucoma.

Effects of multipurpose solutions on the viability and encystment of acanthamoeba determined by flow cytometry.

PURPOSE: To evaluate simultaneously the effects of multipurpose contact lens care solution (MPS) on the viability and encystment of Acanthamoeba using flow cytometry. METHODS: Viability and encystment rate were evaluated using Acanthamoeba castellanii (ATCC 50514 and ATCC 50370) and three clinical strains of Acanthamoeba spp. isolated from patients with Acanthamoeba keratitis. Acanthamoeba trophozoites (1.0 × 10(5) cells/mL) were exposed to four kinds of commercially available MPSs for 24 hours. After dispensing the cell suspension into two portions, one portion was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cysts, and the other portion was stained with a mixture of Congo Red and 3% sarkosyl (CRS), a detergent to lyse the trophozoites and pseudocysts. Flow cytometric analysis of the treated portions was then carried out on an EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites, "pseudocyst") were calculated by the rates of CR-stained, CR-nonstained, and CRS-stained populations, respectively. Ultrastructural features of resistant (mature or immature) cysts and pseudocysts were observed by transmission electron microscopy. RESULTS: Resistant cysts and rounded trophozoites (pseudocysts) were stained with CR, whereas native (unrounded) trophozoites were not. Resistant cysts were also stained with CRS unlike pseudocysts. Three clinical isolates showed higher resistance and higher encystment rates than two ATCC strains when treated with encystment-positive control solution. Disinfecting efficacy of each MPS was not directly related to each encystment rate. Transmission electron microscopy observations showed basic differences in the ultrastructure of pseudocysts produced by MPSs and resistant cysts. CONCLUSIONS: These results suggest that viability and encystment of Acanthamoeba are independent phenomena, and therefore disinfecting efficacy of MPS and encystment rates of Acanthamoeba should be evaluated, respectively. Thus, it is important to evaluate simultaneously the disinfecting efficacies and encystment rates of newly developed premarket MPS using the authors' novel flow cytometric methods.

Dynamic patterns of histone lysine methylation in the developing retina.

PURPOSE: Histone lysine methylation (HKM) is an important epigenetic mechanism that establishes cell-specific gene expression and functions in development. However, epigenetic control of retinal development is poorly understood. To study the roles of HKM in retinogenesis, the authors examined the dynamic changes of three HKM modifications and of two of their regulators, the histone methyltransferases (HMTases) Ezh2 and G9a, in the mouse retina. METHODS: Retinal sections and lysates from embryonic day 16 through adult were processed for immunohistochemistry and immunoblotting using antibodies against various marks and HMTases. To further analyze the biological functions of HKM, the effects of small molecule inhibitors of HMTases were examined in vitro. RESULTS: Methylation marks of trimethyl lysine 4 and 27 on histone H3 (H3K4me3 and H3K27me3) were detected primarily in differentiated retinal neurons in the embryonic and adult retina. In contrast, dimethyl lysine 9 on histone H3 (H3K9me2) was noted in early differentiating retinal ganglion cells but was lost after birth. The HMTases controlling H3K27me3, H3K9me2, Ezh2, and G9a were enriched in the inner embryonic retina during the period of active retinogenesis. Using the chemical inhibitors of Ezh2 and G9a, the authors reveal a role for HKM in regulating retinal neuron survival. CONCLUSIONS: HKM is a dynamic and spatiotemporally regulated process in the developing retina. Epigenetic regulation of gene transcription by Ezh2- and G9a-mediated HKM plays crucial roles in retinal neuron survival and may represent novel epigenetic targets to enhance viability in retinal neurodegenerative diseases such as glaucoma.

Sigma-1 receptor regulation of voltage-gated calcium channels involves a direct interaction.

PURPOSE: The sigma-1 receptor belongs to a recently discovered family of transmembrane proteins expressed in the central nervous system, including the eye, and mediates the regulation of ion channels. The exact function of sigma receptors remains to be elucidated. The purpose of this study was to investigate the effect of sigma-1 receptor ligands on calcium homeostasis in a retinal ganglion cell line (RGC)-5 and rat primary RGCs. METHODS: Calcium imaging was used to assess the effect of sigma-1 receptor agonist (+)-N-allylnormetazocine ((+)-SKF10047) on potassium chloride (KCl)-induced calcium influx in RGC-5. The whole-cell patch clamp technique was used to analyze the effect of (+)-SKF10047 on calcium currents in primary RGCs. Coimmunoprecipitation assessed the interaction between the sigma-1 receptor and the L-type voltage-gated calcium channel. RESULTS: The sigma-1 receptor agonist (+)-SKF10047 inhibited potassium chloride (KCl)-induced calcium influx. The sigma-1 receptor antagonist, BD1047, reversed the inhibitory effect of (+)-SKF10047. Whole-cell patch clamp recordings of rat cultured primary RGCs demonstrated that (+)-SKF10047 inhibited calcium currents. Coimmunoprecipitation studies demonstrated an association between L-type calcium channels and the sigma-1 receptors. CONCLUSIONS: These results suggest that sigma-1 receptor activation can regulate calcium homeostasis and signaling in RGCs, likely by directly influencing the activity of L-type voltage-gated calcium channels. Regulation of calcium influx in RGCs by sigma-1 receptor ligands may represent in part the neuroprotective effect of sigma-1 receptors.

sigma-1 receptors protect RGC-5 cells from apoptosis by regulating intracellular calcium, Bax levels, and caspase-3 activation.

PURPOSE: sigma-1 Receptor ligands prevent neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. However, the molecular mechanism of the neuroprotective effect remains to be elucidated. The present study was undertaken to determine whether sigma-1 receptor agonists provide neuroprotection by decreasing glutamate-induced calcium mobilization and preventing apoptotic gene expression. METHODS: Cell death was measured by using a calcein-AM/propidium iodide cell-survival assay. Western blot analysis determined the expression levels of Bax in normal RGC-5 cells. Caspase-3 activation after glutamate treatment was determined with a carboxyfluorescein caspase-3 detection kit. Glutamate-induced intracellular calcium mobilization was measured by using ratiometric calcium imaging. RESULTS: sigma-1 Receptor-overexpressing RGC-5 (RGC-5-S1R) cells had lower glutamate-induced intracellular calcium mobilization than did normal RGC-5 cells, and the sigma-1 receptor ligand (+)-SKF10047 reduced the glutamate calcium response in normal and RGC-5-S1R cells. (+)-SKF10047 protected RGC-5 cells from glutamate-induced cell death, and the RGC-5-S1R cells showed a significant resistance to glutamate-induced apoptosis compared with the control RGC-5 cells. BD1047, a sigma-1 receptor antagonist, blocked the protective effect of (+)-SKF10047. Western blot analysis showed that (+)-SKF10047 inhibited the increase in Bax after glutamate treatments. Glutamate-mediated cell death involved activation of caspase-3, and sigma-1 receptor activation prevented an increase in caspase-3 expression. CONCLUSIONS: The results suggest that sigma-1 receptors regulate intracellular calcium levels and prevent activation of proapoptotic genes, thus promoting retinal ganglion cell survival. The sigma-1 ligands appear to be neuroprotective and are a potential target for neuroprotective therapeutics.

Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase.

Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).

Characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme for the transfer of mammalian cholesterol from peripheral tissues to the liver. In patients deficient in LCAT, serum cholesterol levels rise and can lead to corneal opacity, proteinuria, anemia, and kidney failure. As early as 1968, relatively low volume transfusion of normal plasma was shown to temporarily correct the abnormal lipoprotein profiles in LCAT-deficient patients. However, despite the cloning, study, and extensive expression of LCAT in mammalian cell lines, there is still no viable, clinical therapy for LCAT deficiency. The current study was initiated to provide a source of recombinant human LCAT for enzyme replacement therapy. Accordingly, human LCAT has been cloned and expressed for the first time in a human cell line. The recombinant LCAT secreted by these cells was purified by phenyl-Sepharose chromatography, analyzed to determine the nature of its glycosylation, and tested for its enzymatic properties. The activity and basic kinetic parameters for the enzyme were determined using both a fluorescent water-soluble substrate and a macromolecular (proteoliposome) substrate. The enzymatic properties and the carbohydrate components of the recombinant LCAT were all sufficiently similar to those of the circulating human plasma enzyme, suggesting that this source of LCAT may be appropriate for use in some form of enzyme replacement therapy.