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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Cell Mol Med. Author manuscript; available in PMC Aug 1, 2012.
Published in final edited form as:
PMCID: PMC3071858
NIHMSID: NIHMS260817

shRNA knockdown of Gucy2e or Cnga1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

Introduction

Photoreceptor cell death is the dominant pathological feature in many retinal diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD) [1,2] [3]. About 36,000 cases worldwide of simplex and familial RP are caused by loss of function mutations in cGMP phosphodiesterase (PDE6) [47]. Although significant advances in our understanding of RP have been made, the exact interplay between defective PDE6 and the onset of RP pathogenesis remains poorly understood.

Several Pde6b mouse models of RP, including Pde6brd1, Pde6brd10, and Pde6bH620Q, have been used to study the mechanisms involved in the disease [8] [9] [10]. Loss of PDE6 enzyme activity has been shown to result in increased levels of cGMP in Pde6brd1 mice [11,12] [1315] and high Ca2+ in Pde6bH620Q mice [8]. It has been hypothesized Pde6b loss of function leads to high cGMP and consequently excessive Ca2+ influx, which is toxic to photoreceptors [8,16,17];[15].

During rod phototransduction, cGMP and Ca2+ are regulated by PDE6, guanylate cyclase (GUCY2), and cGMP-gated Na+/Ca2+ (CNG) channels. The level of cGMP is controlled by opposing activities of PDE6 and GUCY2 and, at sufficiently high levels, CNG channels open to allow increased Ca2+ influx into the rod outer segment (ROS). Thus, during photoexcitation and recovery, changes in cGMP and Ca2+ levels occur in parallel. Light stimulates PDE6 activation, rapid reduction in cGMP, closure of CNG channels and reduced Ca2+ influx. After a light flash, PDE6 is inhibited, and cGMP and Ca2+ are restored to nominal levels by GUCY2 activity and opening of CNG channels, respectively [1836].

Knowledge gained from the Pde6b models has been used for testing different drug and gene therapies [8, Farber, 1994 #2559,3742]. Attempts to prevent or block photoreceptor death using Ca2+ channel blockers has showed limited efficacy. D-cis-diltiazem was tested for the ability to reduce Ca2+ concentrations without affecting cGMP and was reported to prevent the loss of both rods and cones in Pde6rd1 mice [43,44]. However, the extent of protection originally reported has been controversial [4548]. The specificity of D-cis-diltiazem for rod CNG channels has been demonstrated to be lower than L-cis-diltiazem, suggesting this isoform might have a greater effect. However, significant rescue of Pde6b mutant photoreceptors using L-cis-diltiazem is uncertain [4349].

Gene therapy approaches to increase photoreceptor survival has also been employed. The common denominator in these studies is the use of a vector designed to express PDE6β in Pde6b mutant photoreceptors. Different vectors based on adenovirus, adeno-associated virus, lentivirus, simian immunodeficiency virus, and herpes simplex virus, have been tested for their ability to delay degeneration. Although significant morphological and functional restoration has been reported, incomplete long-term rescue was achieved in all cases [8, Farber, 1994 #2559,3841].

In this report, we took an alternative approach to remedy the condition of excess cGMP and Ca2+ in Pde6b mutant mice. We employed lentivirus shRNA technology to knockdown the expression of Gucy2e and Cnga1 to rescue retinal degeneration. We injected viral particles subretinally in newborn (P5) Pde6bH620Q mutant and C57BL/6J mice, measured changes in gene expression, and photoreceptor function and survival. Our results demonstrate this approach is a viable alternative to previously tested pharmacological and gene therapy methods.

Materials and Methods

Mouse lines and husbandry

Mice were used in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology, as well as the Policy for the Use of Animals in Neuroscience Research of the Society for Neuroscience. Pde6bH620Q mice used in this experiment were bred from a colony of mice that has been previously reported [8,50]. All Pde6bH620Q mice analyzed in this study are homozygotes and will be referred to as Pde6bH620Q mutants or Pde6bH620Q mice. Pde6bH620Q and Pde6brd1 strains are in the C3H background and age-matched C57BL/6J (B6) mice were used as controls (The Jackson Laboratory, Bar Harbor, ME).

Histochemical analyses

Mice were sacrificed and H&E retinal sections were obtained as described [19,20,51,52]. The number and morphology of photoreceptors of lentiviral shRNA injected eyes were compared to control eyes. Briefly, quantification of photoreceptor nuclei was conducted on several sections containing the optic nerve as follows: the distance between the optic nerve and the ciliary body was divided into four quadrants and three rows of rows of nuclei were counted within each single quadrant. Averages and standard deviations were calculated from fifteen animals for each time-point. The corneal scar at the 6 o’clock position allowed the identification of the injected inferior retinal half of the sample analyzed. Sectioning proceeded along the long axis of the segment so that each section contained upper and lower retina as well as posterior pole.

Immunoblot analysis

Retinas were homogenized in 10% sodium dodecyl sulfate (SDS) by brief sonication and denatured at 100°C for five minutes. Following centrifugation, total protein content per sample was measured by the DC Protein Assay method (Bio-Rad Laboratories). Proteins were separated by SDS polyacrylamide gel electrophoresis. Samples were then transferred to nitrocellulose membranes, which were blocked in 3% bovine serum albumin (Santa Cruz Biotechnology, Santa Cruz, CA), 150 mmol/L NaCl, 100 mmol/L Tris (pH 7.4), and 0.5% Tween-20 (BSA-TTBS). Membranes were incubated with either rabbit antibody to the GUCY2E (1:500, kindly provided by Alexander Dizhoor), CNGA1 (1:12, kindly provided by Robert Molday), mouse monoclonal IgG1 to rhodopsin (1:500, 1D4, Santa Cruz Biotechnology, sc-57432), or mouse monoclonal IgG2b to α Tubulin (1:500, 6-11B-1, Santa Cruz Biotechnology, sc-23950) antibodies in BSA-TTBS. After washing in TTBS, filters were incubated with either goat anti-rabbit conjugated horseradish peroxidase secondary antibodies (1:10,000, Santa Cruz Biotechnology, sc-2004) or goat anti-mouse IgG- horseradish peroxidase secondary antibodies (1:10,000, Santa Cruz Biotechnology, sc-2005). After washing, antibody complexes were visualized by chemilluminescence detection (Immobilon Western, Millipore Corporation) and Kodak BioMax film (Kodak, Rochester, NY).

Transduction of lentiviral vectors

To knockdown Gucy2e or Cnga1 expression, we injected shRNA lentivirus (1.5 microliter, 2 × 107 ml transducing units (TU) per ml in Dulbecco’s Modified Eagle’s Medium with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin) subretinally into the right eye of Pde6bH620Q mice at P5 (n=75 per gene) and in age-matched control C57BL/6J (n=75). Virus particles were injected at the 6 o’clock position and at 1.5 mm from the limbus, producing a subretinal bubble in mid-periphery retina. The left eye was injected with CMV::EGFP (1.5 microliter, 2 × 107 TU/ml) lentivirus or saline subretinally and used as control. Anesthesia and surgery were performed as described [8].

shRNA vectors deliver a short 21-nucleotide stem hairpin RNA duplexes, designed to decrease the expression of retinal Gucy2e and Cnga1. The lentiviral transduction particles were made from sequence-verified shRNA lentiviral plasmid vectors for mouse genes (SHVRS, Mission® Lentiviral Transduction Particles, Sigma-Aldrich®). Clone sets injected consist of four Gucy2e and five Cnga1 individual clones targeting different regions of the mRNA for each gene. Clone sets used for this analysis are shown in Table 1. Employing different clones allow us to screen for the most effective shRNA for gene knockdown. We expect at least a knockdown efficiency of >70% with one construct from each gene targeted [53].

Table 1
Table shows the shRNA lentiviral plasmids clone sequence, clone ID and titer used to suppress the expression of guanylate cyclase 2e (Gucy2e), (A); and cyclic nucleotide gated channel alpha 1 (Cnga1),(B). TRC, The RNAi Consortium.

Electroretinograms (ERGs)

Visual function was evaluated as described [5459] Electroretinograms (ERGs) were performed weekly from P35 to P90 to assess global retina function in injected and control eyes. We measured ERG b-wave enhancement of rod, maximal and cone responses from shRNA-lentivirus transduced C57BL/6J and Pde6bH620Q mice. Enhancement is defined as the difference in maximum ERG responses of transduced and control fellow eyes, in µV.

Results

The efficiency of shRNA knockdown to ameliorate degeneration was assessed by biochemical, histological, and physiological measurements. Initially, four Gucy2e and five Cnga1 shRNA clones were prescreened for the ability to rescue degeneration. Each clone was injected into the subretinas of a litter of pups. At P56, injected and control retinas were compared histologically and the ability of the clone to rescue degeneration was determined. The most efficient shRNA-Gucy2e and shRNA-Cnga1 clone was selected for further study (Table 1).

Biochemical assessment: Effective and specific knockdown of GUCY2E and CNGA1 expression

The ability of shRNA-Gucy2e and shRNA-Cnga1 viral clones to knockdown protein expression was tested in C57BL/6J mice. Protein levels in retinal lysates were tested by immunoblotting 15 and 60 days post injection. A significant difference in GUCY2E and CNGA1 expression levels was observed between control and shRNA-Gucy2e and shRNA-Cnga1 retinas, respectively (Fig. 1A, B). In contrast, expression of the rod-specific protein, GNAT1, was not significantly different between experimental and control retinas. Furthermore, shRNA-Gucy2e failed to reduce CNGA1 expression. Similarly, shRNA-Cnga1 did not significantly reduce GUCY2E expression.

Fig. 1
Immunoblotting of retinal lysates from P60 wild-type C57BL/6J mice transduced with shRNA-Gucy2e (panel A, +), shRNA-Cnga1 (panel B, +), or shRNA-GFP control lentivirus (panels A and B, −). The antibodies used were anti-GUCY2E, anti-GNAT1, and ...

Histological assessment: C57BL/6J photoreceptors are not detectably affected by shRNA-Gucy2e and shRNA-Cnga1 transduction

To determine if reduction of GUCY2E and CNGA1 expression results in changes in photoreceptor numbers or morphology, sections from shRNA-Gucy2e and shRNA-Cnga1 injected and control C57BL/6J retinas were stained by H&E and compared. Quantification of photoreceptor nuclei was conducted on several sections; retinae transduced by shRNAs have nine rows of photoreceptor nuclei lining areas from the optic nerve to ora serrata, similar to control injected eyes. Ten retinae were analyzed for each viral vector. C57BL/6J shRNA transduced retinae showed no statistically significant differences in retinal structure compared to controls (Table 2). Moreover, neither noticeable morphological changes nor abnormal staining patterns were observed between injected or control retinas (Fig. 2A–D).

Fig. 2
shRNA knockdown has no detectable gross effects on photoreceptor survival or structure. Lentiviral shRNA targeting of Gucy2e (A) and Cnga1 (C) in C57BL/6J mice exhibit identical retinal structure as control retinas (B, D) injected with saline. Eight to ...
Table 2
Quantitative analysis of photoreceptor nuclear rows. The average (AVG) number of photoreceptor nuclei in each transduced eye was counted. The results are shown as means and standard deviation (SD).

Morphological rescue after transduction of Pde6bH620Q photoreceptors after shRNA-mediated knockdown of GUCY2E or CNGA1

Since shRNA knockdown of GUCY2E expression is not associated with gross photoreceptor degeneration in P60 control mice, we tested the ability of the shRNA-Gucy2e vector to rescue Pde6b mutant photoreceptors from degeneration. Lack of outer segments along with a single row of photoreceptor nuclei is typically observed at eight weeks in Pde6bH620Q mutants due to the natural retinal degeneration process. In contrast, Pde6bH620Q retinae transduced by shRNA-Gucy2e showed the number of photoreceptor nuclei was visibly higher than controls (Fig. 3A, B). Pde6bH620Q retinae transduced by shRNA-Gucy2e have on average four rows of photoreceptor nuclei lining areas, whereas Pde6bH620Q retinae transduced by shRNA-GFP or saline exhibited a single row of photoreceptor nuclei without outer segments. Calculated t-tests for total averages in Gucy2e retinas vs. controls gave p-values of 0.0211.

Fig. 3
Lentiviral shRNA targeting of Gucy2e (A) and Cnga1 (C) in Pde6bH620Q mice. At P56, the control retinae (B, D) show a single row of photoreceptors with scattered outer segments, as is typically observed in Pde6bH620Q mutants [Davis, 2008 #4982], (yellow ...

To evaluate whether reducing Cnga1 expression rescued degeneration, we compared histological sections from injected and control Pde6bH620Q eyes at P56. shRNA-Cnga1 injected eyes showed regions containing OS and an average of five rows of photoreceptor nuclei, while control retinas showed a single row of photoreceptor nuclei with scant OS at P56 (Fig. 3C, D).

Differences were quantified in both individual quadrant and total averages (Table 2D). Calculated total averages of Cnga1 transduced vs. control retinas gave very highly statistically significant p-values of 0.0001. Together, these results show photoreceptor survival in Pde6bH620Q mutants is improved for two months after shRNA-Gucy2e or shRNA-Cnga1 transduction.

Functional assessment: Effect of Gucy2e and Cnga1 knockdown on photoreceptor activity in C57BL/6J controls and Pde6bH620Q mice

We first performed ERG analyses to assess the effect of shRNA-Gucy2e or shRNA-Cnga1 transduction on global retinal function from P35 through P90 in C57BL/6J mice (Fig. 4). ERG function was significantly depressed in C57BL/6J controls transduced with shRNA-Gucy2e or shRNA-Cnga1. In contrast, no shRNA-GFP control or saline injected eyes showed ERG depression (Fig. 4, control). Lower a- and b-waves amplitudes were observed at all times tested. The greatest difference among transduced versus control retinas was observed at P60. This difference diminished by P90 (Fig. 4). Dark-adapted maximal ERG depression from C57BL/6J after shRNA-Cnga1 transduction exhibited higher values than shRNA-Gucy2e (Fig. 6).

Fig. 4
Maximal dark-adapted ERG traces of C57BL/6J (wild-type) mice transduced with shRNA-Gucy2e (1) and shRNA-Cnga1 (2), and corresponding control eyes at postnatal days 35, 60 and 90. ERGs were performed on both eyes simultaneously; each color trace represented ...
Fig. 6
ERG b-wave enhancement of rod, maximal and cone responses from shRNA-lentivirus transduced C57BL/6J and Pde6bH620Q mice. The upper row displays C57BL/6J mice aged one to three months transduced with shRNA-Gucy2e (left panel) or shRNA-Cnga1 (right panel). ...

In contrast, maximal ERG responses in Pde6bH620Q eyes after shRNA-Cnga1 and shRNA-Gucy2e transduction exhibited higher values compared to fellow control eyes. shRNA-Gucy2e virus maintained photoreceptor function in Pde6bH620Q mutants for a month. ERGs performed at P35 and P60 showed higher b-wave amplitudes in lentiviral shRNA injected eyes compared to control eyes (Fig. 5, shRNA-Gucy2e). There was preservation of maximal ERG a-wave responses up to 35 days. The largest difference between experimental and control eyes were in maximal mixed rod-cone responses at P35: 105µV vs. 15µV, respectively. However, mice tested at P90 showed extinguished ERGs in both treated and control eyes (Fig. 5, Control).

Fig. 5
Functional rescue of neuronal signaling in Pde6bH620Q retinas transduced with shRNA-Gucy2e (1) and shRNA-Cnga1 (2). Each mutant received a subretinal injection of shRNA-Gucy2e (1) and shRNA-Cnga1 (2) lentivirus in the right eye and saline in the left ...

Retinal function was also improved after one month of injection with shRNA-Cnga1 in Pde6bH620Q mice (Fig. 5). Analysis of electroretinograms showed 75% of eyes (21 of 28) evidenced ERG rescue in responses (Fig. 5; shRNA-Cnga1). There was preservation of maximal ERG a-wave responses up to 60 days. Efficacy of shRNA-Cnga1 is maintained for at least 90 days after injection in Pde6bH620Q mice. In contrast, control injections failed to rescue photoreceptor function (Fig. 5, Control).

Comparison of between vectors show that eyes transduced with shRNA-Cnga1 has higher responses than shRNA-Gucy2e (Fig. 6). Although shRNA-Guyc2e or shRNA-Cnga1 suppressed b-wave amplitudes in C57BL/6J mice, they significantly enhanced b-wave amplitudes in Pde6bH620Q mutants. Maximal ERG b-wave enhancement was detected from P35 to P90 post-transduction (Black solid bars).

To confirm that this effect is not due to non-specific expression of shRNAs or surgical injury response, we also tested mice injected with shRNA-GFP and saline. The ERGs from these mice did not reveal any statistical significant difference between injected eyes and non-injected eyes.

Discussion

We show delivery of shRNAs by lentiviral subretinal transduction resulted in reduction of GUCY2E and CNGA1 expression, and in significant morphological and functional rescue of Pde6bH620Q photoreceptors. These data support the model that high levels of intracellular cGMP and/or Ca2+ plays a role in the PDE6-associated RP.

Our results support cGMP as an initiator of the retinal degeneration process in Pde6b mutant mice. In normal photoreceptors, the concentration of cGMP is controlled by the balance between its synthesis by GUCY2 and its hydrolysis by PDE6 [3742,60]. In Pde6brd1 mice, PDE6 activity is undetectable and cGMP is significantly elevated above controls. In this model, cGMP accumulates to high levels because GUCY2 continue to synthesize cGMP at a basal rate in the absence of PDE6 activity. Since PDE6 and GUCY2 directly catalyze reactions involving cGMP, suppression of GUCY2E expression resulting in increased photoreceptor survival is consistent with cGMP being a primary initiator of PDE6-related retinal degeneration.

However necessary elevated cGMP may be in initiating cell death in these mouse models, other downstream changes are likely to be involved and may be more directly responsible for provoking degeneration. Light-sensitive conductance in photoreceptors is controlled by the effect of cGMP on Ca2+ influx through regulation of CNG channels [6164]. Since abnormal Ca2+ levels has been implicated in causing photoreceptor cell death, toxicity associated with cGMP may be secondary to abnormal regulation of CNG channels[8,16]. Indeed, our data show downregulation of CNG channels by shRNA is associated with increased photoreceptor survival.

To date, success of viral gene therapies to rescue vision has been limited to non-photoreceptor specific diseases such as Rs1, RPE65-related early onset-retinal dystrophy, or photoreceptor diseases involving minimal cell death [6569]. Previous attempts to rescue Pde6b mutant photoreceptors using viral PDE6 expression vectors has had limited success [3741,60]. Although future work is necessary, this approach may not provide enough long-term wild type PDE6 activity to block excess Na+/Ca2+ entry [3842,60]. Our work suggests that downstream interventions may augment this approach. Furthermore, since cGMP and Ca2+ may be a common alteration leading to cell death in other signal-dependent neurodegenerative diseases, future manipulation of cGMP or cation levels with shRNA may allow for the conversion of a progressive degeneration into a stationary disease.

Both RP and the atrophic (dry) form of age-related macular degeneration (AMD) are characterized by an initial loss of rod photoreceptors. AMD manifests clinically by abnormal kinetics in dark adaptation and decreased rod-mediated visual function, followed by the death of cones and the retinal pigment epithelium. Rods are also affected earlier than cones in normal aging. Because reduced PDE6 function is a common denominator in many cases of photoreceptor degenerations, it may be possible to develop treatments based on decreasing cGMP production by GUCY2E and influx of Ca2+/Na+ through CNGA1. Ultimately, the results from these studies will open the possibility of genetic modification in Irish Setters with PDE6B-related degeneration and humans with photoreceptor diseases.

Acknowledgments

Funding

Burroughs-Wellcome Program in Biomedical Sciences Fellow, Charles Culpeper Scholarship, Foundation Fighting Blindness, Hirschl Trust, Schneeweiss Stem Cell Fund, Sylvia Wright Retinal Research Trust, Joel Hoffmann Foundation, Jonas Family Fund, Crowley Research Fund, Jahnigen/Hartford/American Geriatrics Society, Eye Surgery Fund, Bernard Becker-Association of University Professors in Ophthalmology-Research to Prevent Blindness (RPB), and EY018213.

Footnotes

The authors confirm that there are no conflicts of interest.

Contributor Information

Tosi Joaquin, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, Ophthalmology, Pathology & Cell Biology.

Davis Richard, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, Ophthalmology, Pathology & Cell Biology.

Nan-Kai Wang, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, Ophthalmology, Pathology & Cell Biology; Chang Gung Memorial Hospital at Linkou, Department of Ophthalmology; Chang Gung University College of Medicine.

Naumann Matthew, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, Ophthalmology, Pathology & Cell Biology.

Lin Chyuan-Sheng, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, Ophthalmology, Pathology & Cell Biology.

Tsang Stephen, Columbia University, Ophthalmology.

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