PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of hmgLink to Publisher's site
 
Hum Mol Genet. Dec 15, 2009; 18(24): 4770–4780.
Published online Sep 17, 2009. doi:  10.1093/hmg/ddp440
PMCID: PMC2778372
Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism
Xi-Qin Ding,1* Cynthia S. Harry,1 Yumiko Umino,2 Alexander V. Matveev,1 Steven J. Fliesler,3,4,5 and Robert B. Barlow2
1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA,
2The Center for Vision Research, Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY, USA,
3Research Service, Veterans Administration Western New York Healthcare System, University at Buffalo/State University of New York, Buffalo, NY, USA,
4Department of Ophthalmology (Ira G. Ross Eye Institute/Vision Research Center), and
5Department of Biochemistry, University at Buffalo/State University of New York, Buffalo, NY, USA
*To whom correspondence should be addressed at: 940 Stanton L. Young Boulevard, BMSB 553, Oklahoma City, OK 73104, USA. Tel: Phone: +1 4052718001; Fax: +1 4052713548; Email: xi-qin-ding/at/ouhsc.edu
Received July 28, 2009; Accepted September 14, 2009.
The cone cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. This channel is composed of two structurally related subunits, CNGA3 and CNGB3; CNGA3 is the ion-conducting subunit, whereas CNGB3 is a modulatory subunit. Mutations in both subunits are associated with achromatopsia and progressive cone dystrophy, with mutations in CNGB3 alone accounting for 50% of all known cases of achromatopsia. However, the molecular mechanisms underlying cone diseases that result from CNGB3 deficiency are unknown. This study investigated the role of CNGB3 in cones, using CNGB3−/− mice. Cone dysfunction was apparent at the earliest time point examined (post-natal day 30) in CNGB3−/− mice. When compared with wild-type (WT) controls: photopic electroretingraphic (ERG) responses were decreased by ~75%, whereas scotopic ERG responses were unchanged; visual acuity was decreased by ~20%, whereas contrast sensitivity was unchanged; cone density was reduced by ~40%; photoreceptor apoptosis was detected; and outer segment disorganization was observed in some cones. Notably, CNGA3 protein and mRNA levels were significantly decreased in CNGB3−/− mice; in contrast, mRNA levels of S-opsin, Gnat2 and Pde6c were unchanged, relative to WT mice. Hence, we show that loss of CNGB3 reduces biosynthesis of CNGA3 and impairs cone CNG channel function. We suggest that down-regulation of CNGA3 contributes to the pathogenic mechanism by which CNGB3 mutations lead to human cone disease.
Photoreceptor cyclic nucleotide-gated (CNG) channels are localized to the plasma membrane of photoreceptor outer segments (OS) and play a pivotal role in phototransduction (1). In darkness, these channels are held in the open state by cGMP, maintaining an inward current. Via the phototransduction cascade, light induces hydrolysis of cGMP, resulting in closure of the channels and hyperpolarization of the cell. CNG channels are composed of two structurally related subunit types, the A and B subunits. The rod channel consists of CNGA1 and CNGB1 subunits, whereas the cone channel contains CNGA3 and CNGB3 subunits. Heterologous expression studies have shown that the A subunits are responsible for the ion-conducting activity of the channel, whereas the B subunits function as modulators (1,2).
Mutations in the rod CNG channel have been identified in patients with retinitis pigmentosa (3), whereas mutations in the cone CNG channel are associated with achromatopsia, progressive cone dystrophy and early-onset macular degeneration (47). To date, more than 70 mutations have been identified in the human CNGA3 and CNGB3 genes, and mutations in CNGB3 are found in 50% of achromatopsia patients (5). Nevertheless, little is known about the molecular mechanism of retinal pathogenesis resulting from CNGB3 deficiency. The most frequently occurring mutation is the Thr383fsx mutation, which accounts for over 70% of all CNGB3 mutant alleles (5,8,9). It is a frame-shift mutation that results in truncation of the pore-forming loop and the C-terminal cytoplasmic domain; hence, it is a null mutation and no intact CNGB3 is formed. Accordingly, we reasoned that characterization of the retinal phenotype of CNGB3−/− mice might provide valuable insights into understanding the pathogenesis of this mutation.
CNGB3 is a modulatory subunit of the cone CNG channel. Although CNGB3 shares a common topology with CNGA3 and possesses a pore-forming region, expression of CNGB3 alone does not form a functional channel (2). The CNGA3 homomeric channel is fully functional in cultured-cells (2,10), and CNGA3/CNGB3 heteromeric channels display a number of properties typical of native CNG channels. These include ‘flicker’ behavior, increased sensitivity to cAMP and l-cis-diltiazem and reduced sensitivity to blockade by extracellular Ca2+ (2,11), all suggesting that CNGB3 modulates channel physiological properties and function. The high correlation of mutations in CNGB3 with human cone diseases points to an essential role for this protein in cone function. Our recent work showing the interaction between CNGA3 and CNGB3 in the mouse retina (12) also supports this view. Yet, it is unclear why mutations in CNGB3 cause cone defects. Here, we report our recent work to further elucidate the biological role of CNGB3 and mechanisms underlying cone dysfunction. Using CNGB3−/− mice, we show that not only did these mice exhibit electrophysiological defects in their cone photoreceptors, but unexpectedly their CNGA3 protein levels were virtually undetectable and CNGA3 mRNA levels were dramatically reduced compared with wild-type (WT) controls. Since CNGA3 is the ion-conducting subunit of the channel, down-regulation of CNGA3 may represent a major mechanism for cone defects resulting from CNGB3 deficiency.
CNGB3−/− mice were normal in their appearance and body weight up to the oldest age examined (12 months). Up to this age, there was no difference in mortality between WT and knockout mice.
Absence of CNGB3 in CNGB3−/− mice
Absence of CNGB3 in CNGB3−/− mice was confirmed by western blotting, immunofluoresence labeling and RT–PCR. Mice at age of P30 days were used for these detections. Western blot analysis probing with polyclonal anti-CNGB3 antibody detected a moderately intense band in WT retina and a robust band in Nrl−/− (cone-enriched) retina, but no CNGB3-positive band was detected in CNGB3−/− retina (Fig. 1A), as expected. The abundant expression of CNGB3 (and CNGA3) and lack of rod CNG channel in Nrl−/− retina was shown previously (12). Immunofluoresence labeling using the same antibody detected CNGB3 in retinal sections of WT, but not CNGB3−/− mice (Fig. 1B). By RT–PCR, a 242 bp band was amplified from retina of WT, but was absent in CNGB3−/- mice and a 177 bp band for the internal control gene hypoxanthine guanine phosphoribosyl transferase (HPRT) was amplified in both genotypes (Fig. 1C).
Figure 1.
Figure 1.
Absence of CNGB3 in CNGB3−/− mice. (A) Western blot analysis of retinal membrane preparations (probed with polyclonal anti-CNGB3 antibody) showing the absence of CNGB3 in CNGB3−/− mice. Actin was probed as loading control. (more ...)
Impaired cone electroretinographic response in CNGB3−/− mice
Retinal function was evaluated by scotopic and photopic electroretinographic (ERG) recordings in CNGB3−/− mice at P30 days. We found that scotopic responses in CNGB3−/− mice were unchanged (Fig. 2Aa), whereas photopic responses were reduced by ~75% (Fig. 2Ab), relative to WT mice. Heterozygous mice displayed normal scotopic and photopic ERG recordings (data not shown). Serial photopic ERG recordings were performed with varying intensities (ranging from −1.57 to 2.40 log cd s m−2) and varying frequencies (ranging from 1.0 to 3.0 Hz) of light stimuli. These analyses also showed reduced photopic ERG response in CNGB3−/− mice, compared with WT mice. Figure 2B shows representative waveforms of serial photopic ERG recordings at varying intensities (Fig. 2Ba) and varying frequencies (Fig. 2Bb). Hence, ERG analyses reveal a cone defect in CNGB3−/− mice.
Figure 2.
Figure 2.
Decreased cone ERG response in CNGB3−/− mice. (A) Scotopic (a) and photopic (b) ERG analyses were performed in WT and CNGB3−/− mice at P30 days. Photopic ERG responses were decreased by ~75%, whereas scotopic ERG (more ...)
Impaired visual acuity but normal contrast sensitivity of CNGB3−/− mice
Cone function in CNGB3−/− mice was also examined by evaluating their visual acuity and contrast sensitivity. By observing their optomotor responses under photopic conditions, we found the visual acuity to be significantly lower (0.427 ± 0.01 cyc/°) for CNGB3−/− mice than for their litter-mate controls (0.527 ± 0.04 cyc/°) (Fig. 3A). Surprisingly, we found CNGB3−/− mice to have normal contrast sensitivity at lower spatial frequencies, 0.128 cyc/° (Fig. 3B). The reduced visual acuity may reflect the reduced density of functioning cones, we report here while the normal contrast sensitivity may suggest a normal or near normal phototransduction in the remaining cones.
Figure 3.
Figure 3.
Impaired visual acuity but normal contrast sensitivity of CNGB3−/− mice. Visual acuity and contrast sensitivity were measured in WT and CNGB3−/− mice at P90 days by observing their optomotor behavior using two-alternative (more ...)
Disorganization of cone OS in CNGB3−/−mice
Retinal morphology and photoreceptor structure were examined in CNGB3−/− mice at P30 days by light and electron microscopy (EM). No appreciable histological (light microscopic) differences were observed among WT, CNGB3+/− and CNGB3−/− mice (Fig. 4A). Photoreceptor structure was examined by transmission EM. WT and CNGB3+/− mice showed normal ultrastructure of cones and rods (Fig. 4B, left and middle panels), whereas disorganization of OSs of cones, but not the neighboring rods, was observed in CNGB3−/− mice (Fig. 4B, right panel). The observations from transmission EM were further confirmed by immunogold labeling of S-cones using anti-S-opsin antibody. Shorter, abnormal and disorganized cones were detected in CNGB3−/− mice, compared with the more normal appearance of cones seen in retinas from WT and CNGB3+/− mice (Fig. 4C). Hence, lack of CNGB3 alters the structure of cone OSs.
Figure 4.
Figure 4.
Retinal morphology and photoreceptor structure in CNGB3−/− mice. (A) Light microscopic images of WT, CNGB3+/− and CNGB3−/− retinas (P30 days). No appreciable histological differences were observed across the three (more ...)
Cone degeneration of CNGB3−/−mice
We examined whether cones degenerate in CNGB3−/− mice. This was carried out by analyzing cone density on retinal sections that were labeled by lectin peanut agglutinin (PNA) or by anti-S-opsin antibody. Mice at P30 days were used in these analyses. As shown in Figure 5, PNA staining (Fig. 5A) and S-opsin labeling (Fig. 5B) were significantly decreased in CNGB3−/− mice, relative to WT control mice. The correlative quantitative analysis showed that cone density was reduced by ~40% in CNGB3−/− mice (Fig. 5A and B, right panels).
Figure 5.
Figure 5.
Cone degeneration in CNGB3−/− mice. (A and B) Decreased cone density in CNGB3−/− mice, relative to WT mice, detected by PNA staining (A, green) and S-cone opsin labeling (B, red). Shown are confocal fluorescence microscopy (more ...)
We also performed TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) assay to detect apoptosis of photoreceptors in CNGB3−/− mice. The assays were performed using mice at P15 and P30 days. As shown in Figure 5C, there were significantly more TUNEL-positive cells in CNGB3−/− mice, compared with WT mice. The correlative quantitative results showed that the TUNEL-positive cells in CNGB3−/− mice at P15 and P30 days were ~3 and 15 times, respectively, more abundant than that in WT mice (Fig. 5C, right panel). Hence, the decreased cone density and increased TUNEL-positive staining suggest cone degeneration in CNGB3−/− mice.
Decreased expression of CNGA3 protein in CNGB3−/−mice
Expression of CNGA3 was examined by immunofluorescence labeling and western blotting. No CNGA3 immunofluorescence labeling was detected in retinal sections of CNGB3−/− mice (Fig. 6A). With immunoblotting using polyclonal anti-CNGA3 antibody, we detected a very weak CNGA3-positive band in CNGB3−/− retina, a moderately intense band in WT retina and a robust band in Nrl−/− retina (Fig. 6B). Hence, CNGA3 protein was significantly decreased in CNGB3−/− mice.
Figure 6.
Figure 6.
Decreased expression of CNGA3 in CNGB3−/− mice. (A) Confocal immunofluorescence localization of CNGA3 (red) in retinal sections of WT and CNGB3−/− mice at P30 days. No CNGA3 was detected in CNGB3−/− retinal (more ...)
Down-regulation of CNGA3 mRNA in CNGB3−/− mice
As only a small amount of CNGA3 protein was detected in CNGB3−/− mice, quantitative real-time (qRT)-PCR was performed to determine whether CNGA3 mRNA was altered. To our surprise, CNGA3 mRNA was decreased by ~90% in CNGB3−/− retina (Fig. 7Aa). CNGA3 mRNA level in Nrl−/− retinas was ~3-fold higher than that in WT retinas, which is consistent with previous findings showing the abundant expression of CNGA3 in this mouse line (12,13). In qRT-PCR assays, identical results were obtained using three pairs of primers (Table 1) that amplify different regions of the CNGA3 transcript. We then examined whether mRNA levels of other cone-specific protein including S-opsin, cone transducin α-subunit (Gnat2) and cone phosphodiesterase (Pde6c) were altered. As shown in Figure 7Ab–d, mRNA levels of S-opsin, Gnat2 and Pde6c in CNGB3−/− mice were not different from that in WT mice. We also preformed semi-qRT-PCR at varying cycles (15, 20 and 30), and these assays showed similar results. At cycles 15 and 20, CNGA3 mRNA was detected in WT but not in CNGB3−/− mice (Fig. 7B). A weak band was detected in CNGB3−/− mice at cycle 30; at this cycle the mRNA level detected in WT was ~10-fold higher than that in CNGB3−/− mice (Fig. 7B). The presence of CNGA3 gene in CNGB3−/− mice was confirmed by genotyping PCR. Hence, there is a remarkable down-regulation of CNGA3 mRNA in CNGB3−/− mice, and the observed decrease of CNGA3 protein is likely a consequence of the down-regulation of its mRNA.
Figure 7.
Figure 7.
Down-regulation of CNGA3 mRNA in CNGB3−/− mice. (A) qRT-PCR was performed to examine mRNA levels of CNGA3 (a), S-opsin (b), Gnat2 (c) and Pde6c (d) in the retinas of WT, CNGB3−/−and Nrl−/− mice at P30 days. (more ...)
Table 1.
Table 1.
Primers used in this study
This study reveals a cone defect in CNGB3−/− mice and provides the first experimental evidence showing the essential role of CNGB3 in cone function and survival. Impaired cone function in CNGB3−/− mice is evidenced by decreased cone ERG responses and reduced visual acuity. The phenotype of CNGB3−/− mice is similar to, but different from, that in CNGA3−/− mice. CNGB3−/− mice have a residual cone ERG response (~25% of the WT level), whereas CNGA3−/− mice have no recordable cone ERGs (14). As CNGA3 is the ion-conducting subunit, absence of this protein is expected to block cone phototransduction. The residual ERG response in CNGB3−/− mice may be a function of a remaining small amount of CNGA3 homomeric channels. It is known from heterologous expression studies that the homomeric CNGA3 channels are fully functional (10,15,16). In addition, the phenotype of CNGB3−/− mice is similar to that of CNGB1−/− mice. CNGB1 is the modulatory/accessory subunit of the rod CNG channel and olfactory neuron (ORN) CNG channel. CNGB1−/− mice have a residual rod ERG response (~20% of the WT level) (17). It appears that the A subunits can form functional homomeric channels in rod and cone photoreceptors.
The most significant finding of this work is the down-regulation of CNGA3 mRNA in CNGB3−/− mice, suggesting that down-regulation of CNGA3 biosynthesis may be the major mechanism for the reduction of the corresponding protein. In fact, reduction of the CNG channel A subunits has been shown in CNGB1−/− mice. The rod CNG channel subunit CNGA1 was nearly undetectable in CNGB1−/− mice (17); and in a separate study of CNGB1−/− mice, the ORNs CNG channel subunits CNGA2 and CNGA4 were also undetectable in the cilia of ORNs (18). From these studies, CNGB1 was proposed to play a role in the formation and/or OS/cilia transportation of the rod/ORNs CNG channels, and lack of the channels was suggested to be caused by rapid degradation of the channels, as a result of failure of complex formation and OS/cilia targeting (18). Indeed, an impaired surface expression of the channel ion-conducting (A or α) subunits in the absence of the modulatory/accessory (B or β) subunits have been described in other types of ion channels, including the voltage-gated sodium, potassium and calcium channels (19,20). The role of CNGB1 in rod channel transport to rod OS via association with the membrane adaptor protein ankyrin-G was recently described (21). Whether CNGB3 has a similar role in transporting CNGA3 to the plasma membranes of cone OSs and stabilizing the channel complexes remains to be determined. The present work shows a remarkable down-regulation of CNGA3 mRNA in CNGB3−/− mice, suggesting a coordinated, transcriptionally regulated reduction of CNGA3 subunits. This points to a novel role for CNGB3 and shows CNG channel A- and B-subunit expression to be interrelated. Hence, it is likely that the B subunits may not only affect the channel electrophysiological properties, cell surface targeting, stabilization, but also regulate the channel biosynthesis.
At present, the exact mechanism by which CNGA3 mRNA levels are down-regulated in CNGB3−/− mice is unknown. Whether the down-regulation is due to a suppression of transcription or through an enhanced degradation and whether it is CNGB3 itself or CNGA3/CNGB3 complex that regulates CNGA3 gene expression remain to be determined. Apparently, this regulation is photoreceptor-specific, as homomeric CNGA3 channels in heterologous expression system (e.g. in HEK293 cells) can be expressed and are functional. Hence, further studies have to be performed using photoreceptor cells, either in vivo (in the retinas of living animals) or in vitro (cultured photoreceptor cells or retina organ culture). Because cones comprise only 3–5% of the total photoreceptor population in a mouse retina, studying the cellular mechanisms in CNGB3−/− mice (a rod-dominant retina) is challenging. We have shown the cone-dominant Nrl−/− mouse line to be a useful model to study the cone CNG channel, by demonstrating the abundant expression of cone CNG channel and lack of rod CNG channel (12). Thus, the CNGB3−/−/Nrl−/− mouse line may provide a useful model to pursue further studies. Indeed, how CNGB3 regulates CNGA3 biosythesis in cones relates to a new biological question that will require extensive investigation and is beyond the scope of the current study. Using the CNGB3/Nrl double-null mouse line may provide additional insights into how the CNGA3 mRNA is down-regulated. The finding that CNGA3 biosynthesis is reduced in CNGB3−/− mice may have therapeutic significance. Supplementation of CNGB3−/− mice with a CNGA3 transgene or with both CNGA3 and CNGB3 transgenes may help to restore or improve cone function and survival.
Another significant finding from this study came from a behavioral evaluation of CNGB3−/− mice vision showing reduced visual acuity but normal contrast sensitivity. The 20% reduced visual acuity (Fig. 3A) may reflect, in part, the ~40% reduction in functioning cones (Fig. 5). The normal contrast sensitivity suggests a normal or near normal phototransduction in the remaining cones. This observation is similar to that described for cpfl5 mice (a naturally occurring CNGA3 null mutant). Cpfl5 mice exhibit no detectable ERGs but have reduced visual acuity and normal contrast sensitivity (D. Everhart and R. Barlow, unpublished data). However, unlike the preservation of contrast sensitivity in mice lacking components of the CNG channel, deficiency of another phototransduction component, cone transducin α-subunit (Gnat2), in cpfl3 mice leads to a complete loss of cone transduction and cone-mediated vision (22). How cones deficient in CNG channel components can maintain normal contrast sensitivity, whereas those deficient in cone transducin cannot remain to be evaluated further.
CNGB3−/− mice develop cone degeneration. The decreased cone density and increased TUNEL-positive staining is already apparent at P30 days. Cone degeneration is likely attributable to the decreased functional CNG channels, analogous to that in CNGA3−/− mice (14,23). CNGA3−/− mice develop progressive cone degeneration with topographic characteristics; cone degeneration was evident from the second post-natal week on and it proceeded significantly faster in the ventral than in the dorsal part of the retina (23). Whether cone degeneration in CNGB3−/− mice follows a similar time-course and topographic pattern remains to be determined. The molecular pathway coupling deficiency of CNG channel to cone death is unknown. One hypothesis is that a lowered intracellular calcium concentration and accumulation of cGMP, which may occur in a channel-deficient photoreceptor, may trigger oxidative stress and cell apoptotic death (24,25). Cone degeneration in CNGB3−/− mice might also be associated with a potential role of CNGB3 in the OS morphogenesis and integrity, like its rod counterpart CNGB1. It was recently shown that CNGB1 may play a role in the morphogenesis of rod OS discs (26). Our work showing OS disorganization in some cones of CNGB3−/− mice tends to favor this view.
In summary, this study shows a cone defect in CNGB3−/− mice, demonstrating an essential role of CNGB3 in cone function and survival. A remarkable reduction in CNGA3 protein levels along with the down-regulation of its mRNA represents a major mechanism underlying the observed cone defects in CNGB3−/− mice. The down-regulation of CNGA3 mRNA in CNGB3−/− mice demonstrates an essential role of CNGB3 in the biosynthesis of CNGA3. The reduced visual acuity and normal contrast sensitivity of CNGB3−/− mice suggest compensatory mechanisms that maintain cone vision in these mice. In total, these observations contribute significantly to our understanding of the pathogenic mechanism by which CNGB3 mutation leads to human cone disease.
Mice, antibodies and other materials
CNGB3+/− mice (on C57BL/6 N background) were obtained from Deltagen Inc. (San Mateo, CA, USA). The sequence from base 733 to base 749 of mouse CNGB3 mRNA (GenBank accession number AJ243572) was removed by targeting deletion. Cross-mating was performed to obtain homozygous mice. The Nrl−/− mouse line was provided by Dr Anand Swaroop (NEI, Bethesda, MD, USA). WT mice (C57BL/6) were purchased from Charles River Laboratories (Wilmington, MA, USA). All mice were maintained under cyclic light (12 h light–dark) conditions; cage illumination was approximately seven foot-candles during the light cycle. All experiments were approved by the local Institutional Animal Care and Use Committees (Oklahoma City, OK, USA) and conformed to the guidelines on the care and use of animals adopted by the Society for Neuroscience and the Association for Research in Vision and Ophthalmology (Rockville, MD, USA).
The rabbit polyclonal antibodies against mouse CNGA3 and mouse CNGB3 were generated and characterized as described previously (12,15). The rat monoclonal anti-CNGA3 antibody (CNC-7D8) was provided by Dr Benjamin Kaupp (The Institute of Neurosciences and Biophysics, Forschungszentrum, Jülich, Germany). Rabbit polyclonal antibody against mouse S-opsin was provided by Dr Muna Naash (University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA). Affinity purified polyclonal antibody against mouse S-opsin was provided by Dr Sheryl Craft (University of Southern California Keck School of Medicine, Los Angeles, CA, USA). Monoclonal anti-actin antibody was purchased from Abcam, Inc. (Cambridge, MA, USA). Secondary, horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibodies were purchased from Kirkegaard & Perry Laboratories Inc. (Gaithersburg, MD, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), Bio-Rad Laboratories (Hercules, CA, USA) or Invitrogen (Carlsbad, CA, USA).
Scotopic and photopic ERG recordings
ERG testing was carried out as described previously (27). In brief, after a minimum of 4 h dark adaptation, animals were anesthetized by intraperitoneal injection of 85 mg/kg ketamine and 14 mg/kg xylazine. ERG was performed using an LKC system (Gaithersburg, MD, USA) and potentials were recorded using a stainless steel wire contacting the corneal surface through a layer of 2.5% methylcellulose. For assessment of scotopic response, a stimulus intensity of 1.89 log cd s m−2 was presented to the dark-adapted dilated eyes in a Ganzfeld (GS-2000; Nicolet Biomedical, Inc. Madison, WI, USA). To evaluate photopic response, animals were light-adapted for 5 min under a light source of 1.46 log cd s m−2 intensity. Afterward, a strobe flash was presented to the dilated eyes in the Ganzfeld with various intensities (−1.57 to 2.40 log cd s m−2) and frequencies (1.0–3.0 Hz). Responses were differentially amplified, averaged and stored using a Nicolet Compact 4 signal averaging system.
Testing visual acuity and contrast sensitivity
We measured visual acuity of mice by observing their optomotor behavior using a two-alternative forced-choice (2AFC) protocol in combination with a computer-controlled display (OptoMotry©) (28,29). The optomotor stimulus was a vertically oriented sinusoidal pattern (100% contrast) rotated for 5 s periods at a speed of 12°/s under photopic luminance levels (peak-to-trough luminance: 0.36–154.5 cd m−2). The double-blind protocol ensured that the experimenter was unaware of the age, sex and genotype of the tested animal and of the direction of rotation of the sinusoidal pattern presented to the animal. The 2AFC protocol required the observer to choose the direction of pattern rotation based only on the animal's behavior. Using a staircase paradigm, the computer changed grating spatial frequency converging on a threshold of 70% correct observer responses. Each measure of visual acuity is the mean of four trials of each mouse (n = 3–4). Mice were tested under photopic conditions during the first 4 h of their daytime light cycle. Using the same method, we measured contrast sensitivity to a moving sinusoidal pattern having a spatial frequency at the peak of the contrast sensitivity function (0.128 cyc/°) (30). Again the staircase paradigm converged on a threshold of 70% correct observer responses. Contrast sensitivity is the inverse of the grating contrast at threshold.
Light/EM and immunogold labeling
Mouse eye samples were prepared for light/EM as described previously (27,31). Briefly, mouse eyes were enucleated and fixed with 4% formaldehyde (Polysciences, Inc., Warrington, PA, USA) in 0.1 m sodium phosphate buffer, pH 7.4 for 16 h at 4°C. The superior portion of the cornea was marked for orientation prior to enucleation. Fixed eyes were then transferred to PBS or 0.1 m sodium phosphate buffer, pH 7.4, containing 0.02% sodium azide, for storage until processing. Tissue sections were prepared using a Leica microtome (for paraffin sections, 5 µm thickness) and Leica cryostat (for frozen sections, 10 µm thickness), respectively.
For EM and EM immunogold labeling, tissue sections were obtained with a Reichert–Jung Ultracut E microtome using a diamond knife. Thin (600–800 Å) sections were collected on copper 75/300 mesh grids for conventional EM analysis and stained with 2% (w/v) uranyl acetate and Reynolds’ lead citrate. Sections for EM immunogold analysis were collected on nickel 75/300 mesh grids; primary antibody (affinity purified anti-S-opsin) was used at 1:10 dilution; secondary antibody (AuroProbe® 10 nm gold-conjugated goat anti-rabbit IgG, Amersham Biosciences, Pittsburgh, PA, USA) was used at 1:50 dilution. Sections were viewed with a JEOL 100CX electron microscope at an accelerating voltage of 60 keV.
Immunofluorescence labeling and confocal microscopy
Immunofluorescence labelings were performed as described previously (12). Briefly, eye sections were blocked with PBS containing 5% BSA and 0.5% Triton X-100 for 1 h at room temperature. For antigen retrieval eye tissues were incubated in 0.01 m sodium citrate buffer, pH 6.0, for 30 min in a 65°C water bath before blocking. Primary antibody incubation (rat monoclonal anti-CNGA3, 1:50; rabbit polyclonal anti-CNGB3, 1:250 and rabbit polyclonal anti-S-opsin, 1:500) was performed at room temperature for 2 h. Following Alexa- or FITC-conjugated secondary antibody incubation and rinses, slides were mounted and cover-slipped for fluorescence microscope and confocal laser scanning microscope. The fluorescent signals were visualized and images were captured using an Olympus AX70 fluorescence microscope (Olympus Corporation, Center Valley, PA, USA) with the QCapture imaging software (QImaging Corporation, Surrey, BC, Canada) and an Olympus IX81-FV500 confocal laser scanning microscope (Olympus, Melville, NY, USA) with the FluoView imaging software (Olympus).
PNA labeling and cone density evaluation
The lectin PNA is a specific extracellular surface marker of all types of cones. We performed PNA cytochemistry to visualize cones, using the biotinylated PNA (Vector Laboratories, Burlingame, CA, USA) (1:100) and streptavidin-FITC (Sigma-Aldrich). Cone density was assessed as described by Komeima et al. (32), by counting the number of cones present within 0.2 × 0.1 (mm) rectangular areas of retinal sections at four locations of the superior and inferior hemispheres, respectively. The four locations were 33% (S1) and 67% (S2) of the distance between the superior far periphery (retina/ciliary body junction) and the optic nerve and 33% (I1) and 67% (I2) of the distance between the inferior far periphery and the optic nerve.
TUNEL assay
The TUNEL assay was used to study the apoptosis of photoreceptor cells of WT and CNGB3−/− mice. An apoptosis detection kit (ApopTag Plus Peroxidase In Situ Apoptosis Detection; Chemicon, Temecula, CA) and 5 µm thick paraffin-embedded sections were used in this analysis.
Retinal membrane preparation, SDS–PAGE and western blot analysis
Protein SDS–PAGE and western blotting were performed as described previously (12). Briefly, retinas were homogenized in homogenization buffer (10 mm Tris–HCl, pH 7.4, 1 mm EDTA, 200 mm sucrose, 1 mm phenylmethylsulfonyl fluoride). The nuclei and cell debris were removed from the homogenate by centrifugation at 1000g for 10 min at 4°C. The resulting supernatant was centrifuged at 16 000g for 30 min at 4°C. The resultant membranes were used in western blot analysis.
The retinal membrane proteins were subjected to SDS–PAGE and transferred onto polyvinylidene diflouride membranes. Following overnight blocking in 5% non-fat milk at 4°C, blots were incubated with primary antibodies at appropriate dilution ratios (rabbit polyclonal anti-CNGA3, 1:250; rabbit polyclonal anti-CNGB3, 1:250 and mouse monoclonal anti-actin, 1:5000) for 2 h at room temperature. After 3 × 10 min washings with Tris-buffered saline with 0.1% Tween-20, the blots were incubated with HRP-conjugated secondary antibodies (1:12 500) for 1 h at room temperature. SuperSignal® West Dura Extended Duration chemiluminescent substrate (Pierce, Rockford, IL, USA) was used to detect binding of the primary antibodies to their cognate antigens and images were captured using a Kodak Imaging Station (4000 R) (Molecular Imaging, New Haven, CT, USA).
qRT-PCR and semi-qRT-PCR
Total RNA was isolated from mouse retina using Trizol reagent (Invitrogen). Two micrograms of total RNA was reverse-transcribed using an oligo-dT primer and SuperScript III reverse transcriptase (Invitrogen) as per the manufacturer's instructions. Control assays without addition of transcriptase were included and the products were used in the subsequent qRT-PCR as negative controls.
qRT-PCR was performed to detect mRNA levels of CNGA3, S-opsin, Pde6c, Gnat2 and HPRT. Primers were designed to generate amplicons of 180–300 bp using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers for all genes were designed to span introns to avoid amplification from genomic DNA. Table 1 shows primers used in this study. The assays were performed on each cDNA sample using a RT-PCR detection system (iCycler; Bio-Rad Laboratories). A relative gene expression value ΔcT was calculated against HPRT-1 (gene cT−HPRT-1 cT) for each cDNA sample as described (33). Disassociation curve analysis and agarose gel electrophoresis were performed on all PCR products to confirm the proper amplification. The assays were repeated with retinas from at least five animals for each genotype. The semi-qRT-PCR was performed using regular PCR cycling conditions at cycles of 15, 20 and 30. Agarose gel electrophoresis was performed to confirm the proper amplification.
FUNDING
This work was supported by grants from the National Center for Research Resources (P20RR017703), the National Eye Institute [P30EY12190, EY00667 (R.B.B.), EY007361 (S.J.F.)], Lions of Central New York (R.R.B.), the American Health Assistance Foundation (X.Q.D.) and unrestricted grants from Research to Prevent Blindness (R.B.B., S.J.F.).
ACKNOWLEDGEMENT
We thank Drs Anand Swaroop, Benjamin Kaupp, Muna Naash and Sheryl Craft for providing Nrl−/− mice; the monoclonal anti-CNGA3, the polyclonal anti-S opsin and the affinity purified polyclonal anti-S opsin. We thank Barbara Nagel, Carla Hansens and Jianhua Xu for providing excellent technical assistance.
Conflict of Interest statement. None declared.
1. Zagotta W.N., Siegelbaum S.A. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 1996;19:235–263. [PubMed]
2. Gerstner A., Zong X., Hofmann F., Biel M. Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina. J. Neurosci. 2000;20:1324–1332. [PubMed]
3. Dryja T.P., Finn J.T., Peng Y.W., McGee T.L., Berson E.L., Yau K.W. Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc. Natl Acad. Sci. USA. 1995;92:10177–10181. [PubMed]
4. Kohl S., Baumann B., Broghammer M., Jagle H., Sieving P., Kellner U., Spegal R., Anastasi M., Zrenner E., Sharpe L.T., et al. Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21. Hum. Mol. Genet. 2000;9:2107–2116. [PubMed]
5. Kohl S., Varsanyi B., Antunes G.A., Baumann B., Hoyng C.B., Jagle H., Rosenberg T., Kellner U., Lorenz B., Salati R., et al. CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia. Eur. J. Hum. Genet. 2005;13:302–308. [PubMed]
6. Nishiguchi K.M., Sandberg M.A., Gorji N., Berson E.L., Dryja T.P. Cone cGMP-gated channel mutations and clinical findings in patients with achromatopsia, macular degeneration, and other hereditary cone diseases. Hum. Mutat. 2005;25:248–258. [PubMed]
7. Wissinger B., Gamer D., Jagle H., Giorda R., Marx T., Mayer S., Tippmann S., Broghammer M., Jurklies B., Rosenberg T., et al. CNGA3 mutations in hereditary cone photoreceptor disorders. Am. J. Hum. Genet. 2001;69:722–737. [PubMed]
8. Johnson S., Michaelides M., Aligianis I.A., Ainsworth J.R., Mollon J.D., Maher E.R., Moore A.T., Hunt D.M. Achromatopsia caused by novel mutations in both CNGA3 and CNGB3. J. Med. Genet. 2004;41:e20. [PMC free article] [PubMed]
9. Wiszniewski W., Lewis R.A., Lupski J.R. Achromatopsia: the CNGB3 p.T383fsX mutation results from a founder effect and is responsible for the visual phenotype in the original report of uniparental disomy 14. Hum. Genet. 2007;121:433–439. [PubMed]
10. Faillace M.P., Bernabeu R.O., Korenbrot J.I. Cellular processing of cone photoreceptor cyclic GMP-gated ion channels: a role for the S4 structural motif. J. Biol. Chem. 2004;279:22643–22653. [PubMed]
11. Peng C., Rich E.D., Varnum M.D. Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels. J. Biol. Chem. 2003;278:34533–34540. [PubMed]
12. Matveev A.V., Quiambao A.B., Browning Fitzgerald J., Ding X.Q. Native cone photoreceptor cyclic nucleotide-gated channel is a heterotetrameric complex comprising both CNGA3 and CNGB3: a study using the cone-dominant retina of Nrl−/− mice. J. Neurochem. 2008;106:2042–2055. [PMC free article] [PubMed]
13. Farjo R., Skaggs J.S., Nagel B.A., Quiambao A.B., Nash Z.A., Fliesler S.J., Naash M.I. Retention of function without normal disc morphogenesis occurs in cone but not rod photoreceptors. J. Cell Biol. 2006;173:59–68. [PMC free article] [PubMed]
14. Biel M., Seeliger M., Pfeifer A., Kohler K., Gerstner A., Ludwig A., Jaissle G., Fauser S., Zrenner E., Hofmann F. Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3. Proc. Natl Acad. Sci. USA. 1999;96:7553–7557. [PubMed]
15. Ding X.Q., Fitzgerald J.B., Matveev A.V., McClellan M.E., Elliott M.H. Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains. Biochemistry. 2008;47:3677–3687. [PubMed]
16. Yu W.P., Grunwald M.E., Yau K.W. Molecular cloning, functional expression and chromosomal localization of a human homolog of the cyclic nucleotide-gated ion channel of retinal cone photoreceptors. FEBS Lett. 1996;393:211–215. [PubMed]
17. Huttl S., Michalakis S., Seeliger M., Luo D.G., Acar N., Geiger H., Hudl K., Mader R., Haverkamp S., Moser M., et al. Impaired channel targeting and retinal degeneration in mice lacking the cyclic nucleotide-gated channel subunit CNGB1. J. Neurosci. 2005;25:130–138. [PMC free article] [PubMed]
18. Michalakis S., Reisert J., Geiger H., Wetzel C., Zong X., Bradley J., Spehr M., Huttl S., Gerstner A., Pfeifer A., et al. Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping. J. Biol. Chem. 2006;281:35156–35166. [PMC free article] [PubMed]
19. Campomanes C.R., Carroll K.I., Manganas L.N., Hershberger M.E., Gong B., Antonucci D.E., Rhodes K.J., Trimmer J.S. Kv beta subunit oxidoreductase activity and Kv1 potassium channel trafficking. J. Biol. Chem. 2002;277:8298–8305. [PubMed]
20. Manganas L.N., Trimmer J.S. Subunit composition determines Kv1 potassium channel surface expression. J. Biol. Chem. 2000;275:29685–29693. [PubMed]
21. Kizhatil K., Baker S.A., Arshavsky V.Y., Bennett V. Ankyrin-G promotes cyclic nucleotide-gated channel transport to rod photoreceptor sensory cilia. Science. 2009;323:1614–1617. [PMC free article] [PubMed]
22. Alexander J.J., Umino Y., Everhart D., Chang B., Min S.H., Li Q., Timmers A.M., Hawes N.L., Pang J.J., Barlow R.B., et al. Restoration of cone vision in a mouse model of achromatopsia. Nat. Med. 2007;13:685–687. [PubMed]
23. Michalakis S., Geiger H., Haverkamp S., Hofmann F., Gerstner A., Biel M. Impaired opsin targeting and cone photoreceptor migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. Invest. Ophthalmol. Vis. Sci. 2005;46:1516–1524. [PubMed]
24. Fain G.L. Why photoreceptors die (and why they don't) Bioessays. 2006;28:344–354. [PubMed]
25. Pierce E.A. Pathways to photoreceptor cell death in inherited retinal degenerations. Bioessays. 2001;23:605–618. [PubMed]
26. Zhang Y., Molday L.L., Molday R.S., Sarfare S.S., Woodruff M.L., Fain G.L., Kraft T.W., Pittler S.J. Knockout of GARPs and the beta-subunit of the rod cGMP-gated channel disrupts disk morphogenesis and rod outer segment structural integrity. J. Cell Sci. 2009;122:1192–1200. [PubMed]
27. Ding X.Q., Nour M., Ritter L.M., Goldberg A.F., Fliesler S.J., Naash M.I. The R172W mutation in peripherin/rds causes a cone-rod dystrophy in transgenic mice. Hum. Mol. Genet. 2004;13:2075–2087. [PubMed]
28. Prusky G.T., Douglas R.M. Characterization of mouse cortical spatial vision. Vision Res. 2004;44:3411–3418. [PubMed]
29. Umino Y., Everhart D., Solessio E., Cusato K., Pan J.C., Nguyen T.H., Brown E.T., Hafler R., Frio B.A., Knox B.E., et al. Hypoglycemia leads to age-related loss of vision. Proc. Natl Acad. Sci. USA. 2006;103:19541–19545. [PubMed]
30. Umino Y., Solessio E., Barlow R.B. Speed, spatial, and temporal tuning of rod and cone vision in mouse. J. Neurosci. 2008;28:189–198. [PMC free article] [PubMed]
31. Chakraborty D., Ding X.Q., Conley S.M., Fliesler S.J., Naash M.I. Differential requirements for retinal degeneration slow intermolecular disulfide-linked oligomerization in rods versus cones. Hum. Mol. Genet. 2009;18:797–808. [PMC free article] [PubMed]
32. Komeima K., Rogers B.S., Lu L., Campochiaro P.A. Antioxidants reduce cone cell death in a model of retinitis pigmentosa. Proc. Natl Acad. Sci. USA. 2006;103:11300–11305. [PubMed]
33. Farjo R., Skaggs J., Quiambao A.B., Cooper M.J., Naash M.I. Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS ONE. 2006;1:e38. [PMC free article] [PubMed]
Articles from Human Molecular Genetics are provided here courtesy of
Oxford University Press