Chyuan-Sheng Lin

Functional Rescue of Degenerating Photoreceptors in Mice Homozygous for a Hypomorphic cGMP Phosphodiesterase 6 b Allele (Pde6bH620Q)

PURPOSE Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6alpha and PDE6beta) and two regulatory (PDE6gamma) subunits. In mice homozygous for a nonsense Pde6b(rd1) allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6b(rd1) phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6b(H620Q) was characterized further. METHODS Photoreceptor degeneration in Pde6b(H620Q) homozygotes was documented by histochemistry, whereas PDE6beta expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca(2+) recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6b(H620Q) homozygotes. RESULTS Within 3 weeks of birth, the Pde6b(H620Q) homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6beta expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6b(H620Q) homozygotes exhibited depressed rod function and elevated outer segment Ca(2+). Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors. CONCLUSIONS Pde6b(H620Q) homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca(2+) to promote photoreceptor death. As degeneration in Pde6b(H620Q) mutants is slower than in Pde6b(rd1) mice and can be suppressed by Pde6b transduction, this Pde6b(H620Q) model may provide an alternate means to explore new treatments of RP.

Cellular Origin of Fundus Autofluorescence in Patients and Mice with Defective NR2E3 Gene

Aim: To characterise new clinical features in a family with enhanced S-cone syndrome (ESCS) and investigate the pathogenesis of these clinical features in the homozygous Nr2e3rd7 (rd7) mutant mice. Methods: Four patients from an affected family were included for genotypic and phenotypic study. Eye tissues from rd7 mice were used to detect a possible relationship between macrophages and autofluorescent material by immunohistochemistry (IHC) staining. Results: Homozygous mutation in R311Q in NR2E3 was detected in this family. Colour photographs revealed that white dots do not correlate to hyperautofluorescent spots seen in autofluorescence imaging of the macula. OCT showed rosette-like lesions similar to those found in rd7 mice histology sections. From IHC analysis, we observed that F4/80 (a pan macrophage marker) and autofluorescence were colocalised to the same cells within the retina rosettes. Conclusions: The retinal structure of a young ESCS patient with homozygous R311Q mutation in the NR2E3 gene is similar to that seen in the rd7 mice. The macrophages were found to contain autofluorescent materials in the retinal rosettes of rd7 mice. These data are consistent with macrophage infiltration contributing to the hyperautofluorescent spots found in our patients.

Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.

Mutations in Pde6b lead to high levels of signaling molecules cyclic guanosine monophosphate (cGMP) and Ca2+, which ultimately result in photoreceptor cell death in certain forms of retinitis pigmentosa (RP). The level of cGMP, which is controlled by opposing activities of guanylate cyclase (GUCY) and photoreceptor phosphodiesterase-6 (PDE6), regulates the opening of cyclic nucleotide-gated ion channels [CNG] and thereby controls Ca2+ influx into the outer segments. Using a lentiviral gene therapy approach, we have previously shown that degeneration can be temporarily slowed either by introducing wild-type PDE6β or knocking down expression of GUCY2E and CNGA1 in photoreceptors of Pde6b H620Q , a mouse model for RP. Rescue was transient with either approach. Therefore, we tested a novel combination therapy using bipartite lentiviral vectors designed to both introduce wild-type PDE6β expression and knockdown GUCY2E or CNGA1. Immunoblot analysis shows simultaneous increases in PDE6β and decreases in GUCY2E or CNGA1 in retinas transduced by the vectors, indicating successful transduction. In Pde6b H620Q mutants, we observe rescue of photoreceptor function and an increase in photoreceptor rows as compared with untreated controls. However, no evidence of prolonged rescue beyond the limit of the previously tested single therapy was observed.

shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa.

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca2+) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na+ and Ca2+ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na+ and Ca2+ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca2+ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca2+ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6bH620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6bH620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.

Therapeutic Margins in a Novel Preclinical Model of Retinitis Pigmentosa

The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, Pde6bStop, which allows us to temporally correct PDE6-deficiency. Whereas untreated mutants exhibit degeneration, activation of Cre-loxP recombination in early embryogenesis produced stable long-term rescue. Reversal at later time-points showed partial long-term or short-lived rescue. Our results suggest stable restoration of retinal function by gene therapy can be achieved if a sufficient number of rods are treated. Because patients are generally diagnosed after extensive loss of rods, the success of clinical trials may depend on identifying patients as early as possible to maximize the number of treatable rods.

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.

The positive role of the carboxyl terminus of the γ subunit of retinal cGMP-phosphodiesterase in maintaining phosphodiesterase activity in vivo☆

The inhibitory rod cyclic GMP-phosphodiesterase gamma subunit, PDEgamma, is a key component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice that lack PDEgamma due to a targeted disruption of the gene encoding PDEgamma, (Pdeg) suffer from a very rapid and severe photoreceptor degeneration. Previously, deletions in the carboxyl-terminal domain of PDEgamma blocked its ability to inhibit trypsin-activated PDE activity, in vitro. In other words, these mutations eliminated PDEgammas control on the catalytic activity of PDEalpha and PDEbeta. To study the in vivo effects resulting from the deletion of the last seven amino acids of the PDEgamma carboxyl terminal, this PDEgamma allele (Del7C) was introduced as a transgene Pdeg(tm1)/Pdeg(tm1) mice. These animals could only synthesize transgenic mutant PDEgamma. The mutant retinas were expected to display a higher basal level of PDE activity and lower cGMP levels in light and darkness than the PDEgamma knockout mice, which would allow the rescue of their photoreceptors. Instead, our results showed that the Del7C transgene could not complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. In fact, animals carrying the Del7C transgene have low PDE activity as well as reduced PDEalpha and PDEbeta content.

Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J, a Model of Glaucoma and Other Complex Diseases

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.

Phenotype-genotype correlations in autosomal dominant retinitis pigmentosa caused by RHO, D190N

Purpose: To phenotype a family with RHO (Asp190Asn or D190N) dominantly inherited retinitis pigmentosa (RP) and to describe an approach to surveying affected families. Methods: Four patients from a family with a history of autosomal dominant RP had complete clinical examinations and underwent full-field electroretinography (ERG), fundus autofluorescence (AF) imaging, and genetic testing. One patient had microperimetry (MP) mapping. Results: The patients’ ages ranged from 6 years to 47 years. The proband, the father, had fundoscopic findings typical of RP. A small hyperfluorescent ring centered at the fovea was apparent on AF. MP showed preservation of central 7 degrees of visual field within this ring. The three children were all asymptomatic with visual acuity of 20/15 in each eye. One child had mild retinal pigment epithelium migration on fundoscopy; the other two children had normal fundoscopic examinations. Two children showed increased parafoveal AF. In the two affected children, average ERG b-wave implicit times were delayed in scotopic conditions, and maximal ERG tracings had abnormal waveforms. Genetic analysis confirmed that two of three asymptomatic children carried the D190N allele. Conclusions: Patients with RHO (D190N) autosomal dominant retinitis pigmentosa (adRP) can show classic signs of RP on fundus examination and may be able to maintain good central visual acuity into adulthood. By combining clinical examination with AF imaging and electrophysiology, it is possible to offer presymptomatic clinical evaluation to families with this RP.

CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.