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Identification of a new mutant allele, Grm6nob7, for complete congenital stationary night blindness

Published online by Cambridge University Press:  11 May 2015

HAOHUA QIAN ,
RUI JI ,
RONALD G. GREGG  and
NEAL S. PEACHEY
HAOHUA QIAN
Affiliation:
Visual Function Core, National Eye Institute, National Institutes of Health, Bethesda, Maryland
RUI JI
Affiliation:
Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, Kentucky
RONALD G. GREGG
Affiliation:
Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, Kentucky Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky
NEAL S. PEACHEY*
Affiliation:
Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio
*
*Address correspondence to: NealPeachey, Cole Eye Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH44195. E-mail: peachen@ccf.org
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Abstract

Electroretinogram (ERG) studies identified a new mouse line with a normal a-wavebut lacking the b-wave component. The ERG phenotype of this new allele,nob7, matched closely that of mouse mutants forGrm6, Lrit3, Trpm1, andNyx, which encode for proteins expressed in depolarizingbipolar cells (DBCs). To identify the underlying mutation, we first crossednob7 mice with Grm6nob3 mutants and measured the ERGs in offspring. All the offspring lacked theb-wave, indicating that nob7 is a new allele forGrm6: Grm6nob7. Sequence analyses of Grm6nob7 cDNAs identified a 28 base pair insertion between exons 8 and 9, whichwould result in a frameshift mutation in the open reading frame that encodes themetabotropic glutamate receptor 6 (Grm6). Sequencing both thecDNA and genomic DNA from exon 8 and intron 8, respectively, from theGrm6nob7 mouse revealed a G to A transition at the last position in exon 8. Thismutation disrupts splicing and the normal exon 8 is extended by 28 base pairs,because splicing occurs 28 base pairs downstream at a cryptic splice donor.Consistent with the impact of the resulting frameshift mutation, there is a lossof mGluR6 protein (encoded by Grm6) from the dendritic tips ofDBCs in the Grm6nob7 retina. These results indicate that Grm6nob7 is a new model of the complete form of congenital stationary nightblindness, a human condition that has been linked to mutations ofGRM6.

Keywords

Congenital stationary night blindnessElectroretinogramMetabotropic glutamate receptor 6

Information

Type
Research Article
Information
Visual Neuroscience , Volume 32 , 2015 , E004
Copyright
Copyright © Cambridge University Press 2015 

Introduction

Mutations in GRM6, which encodes the metabotropic glutamate receptor 6 (mGluR6), underlie one form of complete congenital stationary night blindness (cCSNB; Dryja et al., Reference Dryja, McGee, Berson, Fishman, Sandberg, Alexander, Derlacki and Rajagopalan2005; Zeitz et al., Reference Zeitz, van Genderen, Neidhardt, Luhmann, Hoeben, Forster, Wycisk, Mátyás, Hoyng, Riemslag, Meire, Cremers and Berger2005). While rod-mediated vision is severely impaired in patients with cCSNB due to GRM6 mutations, their deficits in visual acuity or other measures of cone-mediated vision are more variable and may be normal (Dryja et al., Reference Dryja, McGee, Berson, Fishman, Sandberg, Alexander, Derlacki and Rajagopalan2005; Zeitz et al., Reference Zeitz, van Genderen, Neidhardt, Luhmann, Hoeben, Forster, Wycisk, Mátyás, Hoyng, Riemslag, Meire, Cremers and Berger2005; Godara et al., Reference Godara, Cooper, Sergouniotis, Diederichs, Streb, Genead, McAnany, Webster, Moore, Dubis, Neitz, Dubra, Stone, Fishman, Han, Michaelides and Carroll2012; Sergouniotis et al., Reference Sergouniotis, Robson, Li, Devery, Holder, Moore and Webster2012; Bijveld et al., Reference Bijveld, Florijn, Bergen, van den Born, Kamermans, Prick, Riemslag, van Schooneveld, Kappers and van Genderen2013). mGluR6 is the glutamate receptor used by depolarizing bipolar cells (DBCs; Masu et al., Reference Masu, Iwakabe, Tagawa, Miyoshi, Yamashita, Fukuda, Sasaki, Hiroi, Nakamura, Shigemoto, Takada, Nakamura, Nakao, Katsuki and Nakanishi1995), and GRM6 mutations impair vision when DBCs can no longer modulate transient receptor potential melastatin 1 (TRPM1) channel activity in response to changes in extracellular glutamate (Morgans et al., Reference Morgans, Brown and Duvoisin2010).

Our understanding of the impact of abnormal mGluR6 activity on retinal function has been expanding through the availability of mouse Grm6 mutants. Masu et al. (Reference Masu, Iwakabe, Tagawa, Miyoshi, Yamashita, Fukuda, Sasaki, Hiroi, Nakamura, Shigemoto, Takada, Nakamura, Nakao, Katsuki and Nakanishi1995) characterized the retina of Grm6 tmNak/tmNak mice, referred to here as Grm6 −/−. They noted that retinal structure was intact, despite the loss of the electroretinogram (ERG) b-wave component, which reflects DBC activity (Kofuji et al., Reference Kofuji, Ceelen, Zahs, Surbeck, Lester and Newman2000). Two additional mutants were discovered when no b-wave (nob) mice were identified during screening of mice generated in a N-ethyl-N-nitrosourea mutagenesis program (Grm6 nob3; Pinto et al., Reference Pinto, Vitaterna, Shimomura, Siepka, Balannik, McDearmon, Omura, Lumayag, Invergo, Glawe, Cantrell, Inayat, Olvera, Vessey, McCall, Maddox, Morgans, Young, Pletcher, Mullins, Troy and Takahashi2007) or mouse colonies at the Jackson Laboratory (Grm6 nob4; Maddox et al., Reference Maddox, Vessey, Yarbrough, Invergo, Cantrell, Inayat, Balannik, Hicks, Hawes, Byers, Smith, Hurd, Howell, Gregg, Chang, Naggert, Troy, Pinto, Nishina and McCall2008). While normal retinal structure was observed in both Grm6 nob3 (Pinto et al., Reference Pinto, Vitaterna, Shimomura, Siepka, Balannik, McDearmon, Omura, Lumayag, Invergo, Glawe, Cantrell, Inayat, Olvera, Vessey, McCall, Maddox, Morgans, Young, Pletcher, Mullins, Troy and Takahashi2007) and Grm6 nob4 mice (Maddox et al., Reference Maddox, Vessey, Yarbrough, Invergo, Cantrell, Inayat, Balannik, Hicks, Hawes, Byers, Smith, Hurd, Howell, Gregg, Chang, Naggert, Troy, Pinto, Nishina and McCall2008), differences in receptive field abnormalities of retinal ganglion cells (RGCs) between the two mutants were noted. These studies indicate the value of mouse models of night-blinding disorders and of allelic series for further understanding human disease. Here, we report the discovery and initial characterization of a new nob mouse mutant (nob7), which involves a novel spontaneous mutation in Grm6.

Materials and methods

Mice

All procedures used in animal experiments were approved by the Institutional Animal Care & Use Committees of the institutions involved and conform to the principles regarding the care and use of animals adopted by the American Physiological Society and the Society for Neuroscience. Homozygous nob7 mice were identified in the course of ERG studies of C57BL/6J mice conducted at the National Eye Institute. After establishing that nob7 was inherited as an autosomal recessive trait, affected mice were transferred to Cleveland Clinic for further analysis including a cross to Grm6 nob3 (nob3) mice (Maddox et al., Reference Maddox, Vessey, Yarbrough, Invergo, Cantrell, Inayat, Balannik, Hicks, Hawes, Byers, Smith, Hurd, Howell, Gregg, Chang, Naggert, Troy, Pinto, Nishina and McCall2008). C57BL/6J mice were used as controls.

Electroretinography

After overnight dark adaptation, mice were anesthetized (ketamine: 80 mg/kg; xylazine: 16 mg/kg), their pupils were dilated (1% tropicamide and 2.5% phenylephrine HCl eyedrops) and the corneal surface was anesthetized (1% proparacaine HCl). ERGs were recorded from the corneal surface using a stainless steel wire. Needle electrodes placed in the cheek and tail served as reference and ground leads, respectively. Responses to strobe light flashes were amplified (0.03–1000 Hz) and stored using an LKC (Gaithersburg, MD) UTAS E-3000 signal averaging system. ERGs were recorded under stimulus conditions that allow the response properties of rod- and cone-driven components to be defined using published protocols (Gregg et al., Reference Gregg, Kamermans, Klooster, Lukasiewicz, Peachey, Vessey and McCall2007).

Sequencing

Total RNA was isolated from retinas using TrIzol reagent as per the manufacturer's protocol (Life Technologies, Carlsbad, CA). cDNA was synthesized from total RNA using the Superscript II reverse transcriptase as per the manufacturer's protocol (GE Biosystems). The entire coding region of Grm6 was amplified with mGrm6-ex01F (5′-CAATGTCTTGCGCCTGTTTG-3′) and mGrm6-ex10R (5′-GTGGAGGTCTTCTTGAGGCT-3′), which spanned the open reading frame encoded by exons 2–10. The PCR fragment was cloned and then sequenced on an Applied Biosystems (Carlsbad, CA) 3130XL Sequencer (using a 50 cm array and POP7 polymer). The causative mutation was confirmed to be present in genomic DNA.

Immunohistochemistry

Dissected retinas were immersion fixed for 15 min in 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), then washed in PB, cryoprotected through a graded sucrose series, and frozen in OCT (Sakura Finetek, Torrence, CA):20% sucrose (2:1; Barthel & Raymond, Reference Barthel and Raymond1990). 16-μm sections were cut on a cryostat, mounted onto Super-Frost glass slides, air-dried, and stored at −80°C.

Sections were brought to room temperature, washed in PB saline (PBS) for 5 min, PBX (PBS containing 0.5% (v/v) Triton X-100) for 5 min, and then incubated in a blocking solution (PBX containing 5% (v/v) normal goat serum) for 1 h. Primary antibodies were diluted in the blocking solution and incubated on retinal sections at room temperature overnight. Primary antibodies and dilutions used were: anti-mGluR6 (1:1000; Cao et al., Reference Cao, Posokhova and Martemyanov2011) and anti-CACNA1F (1:1000; BD Biosciences). After incubation with the primary antibody, sections were washed three times in PBS for 5 min each, and subsequently incubated with fluorescently labeled secondary antibodies (1:1000 in blocking solution) at room temperature for 1 h. Secondary antibodies were Alexa 488 goat anti-rabbit and Alexa 555 goat anti-mouse (Invitrogen, Carlsbad, CA). Slides were then washed three times in PBS and coverslipped with Immunomount (ThermoShandon, Pittsburgh, PA). Sections were imaged on an Olympus (Center Valley, PA) FV1000 confocal microscope, using a 60× oil objective (1.45 NA). Images shown are maximum projections of confocal stacks, adjusted for contrast and brightness with Fluoview software.

Results and discussion

The nob7 mutant was discovered during ERG testing of mice anticipated to be wild type. Fig. 1 compares representative ERGs obtained from control and nob7 littermates. Under dark-adapted conditions (Fig. 1A, left), the control response is dominated by the cornea positive b-wave. In response to high luminance stimuli, the b-wave is preceded by the negative polarity a-wave, reflecting the light-induced closure of cation channels along rod photoreceptor outer segments (Penn & Hagins, Reference Penn and Hagins1969). In comparison, the b-wave component is absent from responses of nob7 mice, and the a-wave is followed by a negative polarity component, slow PIII, that is generated by Kir4.1 channel activity in Müller glial cells (Kofuji et al., Reference Kofuji, Ceelen, Zahs, Surbeck, Lester and Newman2000; Wu et al., Reference Wu, Marmorstein, Kofuji and Peachey2004) and is normally masked by the larger amplitude b-wave (Samuels et al., Reference Samuels, Sturgill, Grossman, Rayborn, Hollyfield and Peachey2010).

Fig. 1.

ERG characteristics of nob7 mice. (A) ERGs of nob7 mice lack the b-wave under dark-adapted (left) and light-adapted (right) conditions. (B) ERG waveforms of Grm6 nob7 and Grm6 nob3 mice have a comparable loss of the b-wave component. (C) Representative ERGs from two Grm6 nob3/nob7 double heterozygotes.

Control and nob7 ERGs also differ under light-adapted conditions (Fig. 1A, right). Under these conditions, the ERG represents activity of the cone pathway (Peachey et al., Reference Peachey, Goto, al-Ubaidi and Naash1993). The control cone ERG is dominated by the positive polarity b-wave and high frequency oscillatory potentials, both of which reflect activity through the DBC-pathway (Sharma et al., Reference Sharma, Ball and Peachey2005; Shirato et al., Reference Shirato, Maeda, Miura and Frishman2008). In contrast, cone ERGs of nob7 mice are electronegative.

The nob7 ERG phenotype is inherited in an autosomal recessive fashion and is distinct from that of mouse models for proteins involved in presynaptic aspects of photoreceptor-to-bipolar synaptic transmission, where the b-wave is reduced but still present (Pardue & Peachey, Reference Pardue and Peachey2014). Instead, the nob7 ERG phenotype closely matches that of mouse mutants for Trpm1 (Morgans et al., Reference Morgans, Zhang, Jeffrey, Nelson, Burke, Duvoisin and Brown2009; Koike et al., Reference Koike, Obara, Uriu, Numata, Sanuki, Miyata, Koyasu, Ueno, Funabiki, Tani, Ueda, Kondo, Mori, Tachibana and Furukawa2010; Peachey et al., Reference Peachey, Pearring, Bojang, Hirschtritt, Sturgill-Short, Ray, Furukawa, Koike, Goldberg, Shen, McCall, Nawy, Nishina and Gregg2012a), Nyx (Pardue et al., Reference Pardue, McCall, LaVail, Gregg and Peachey1998), Grm6 (Masu et al., Reference Masu, Iwakabe, Tagawa, Miyoshi, Yamashita, Fukuda, Sasaki, Hiroi, Nakamura, Shigemoto, Takada, Nakamura, Nakao, Katsuki and Nakanishi1995; Pinto et al., Reference Pinto, Vitaterna, Shimomura, Siepka, Balannik, McDearmon, Omura, Lumayag, Invergo, Glawe, Cantrell, Inayat, Olvera, Vessey, McCall, Maddox, Morgans, Young, Pletcher, Mullins, Troy and Takahashi2007; Maddox et al., Reference Maddox, Vessey, Yarbrough, Invergo, Cantrell, Inayat, Balannik, Hicks, Hawes, Byers, Smith, Hurd, Howell, Gregg, Chang, Naggert, Troy, Pinto, Nishina and McCall2008), Gpr179 (Peachey et al., Reference Peachey, Ray, Florijn, Rowe, Sjoerdsma, Contreras-Alcantara, Baba, Tosini, Pozdeyev, Iuvone, Bojang, Pearring, Simonsz, van Genderen, Birch, Traboulsi, Dorfman, Lopez, Ren, Goldberg, Nishina, Lachapelle, McCall, Koenekoop, Bergen, Kamermans and Gregg2012b), and Lrit3 (Neuillé et al., Reference Neuillé, El Shamieh, Orhan, Michiels, Antonio, Lancelot, Condroyer, Bujakowska, Poch, Sahel, Audo and Zeitz2014). Fig. 1B compares the responses obtained from nob7 and nob3 mice. There is an excellent agreement between the responses of the two mutants under both dark- (left) and light-adapted (right) conditions.

To identify the nob7 gene defect, we focused initially on genes expressed in DBCs. Given the close agreement between the ERG phenotypes of nob7 and nob3 mice, we crossed affected animals and examined the ERGs of compound heterozygote offspring. The ERG phenotype of nob3/nob7 compound heterozygous mice was indistinguishable from either parental line (compare Figs. 1C and 1B). This result demonstrates that the nob7 phenotype is caused by a mutation in the Grm6 gene, and the allele will be hereafter referred to as Grm6 nob7.

To identify the Grm6 nob7 mutation, we used reverse transcription PCR to clone and sequence the cDNA fragments representing the open reading frame for mGluR6, from RNA isolated from control and Grm6 nob7 retinas. Sequence analyses of the cDNA clones from the Grm6 nob7 retinas (Fig. 2) identified an insertion between exons 8 and 9 of the normal transcript (ENSEMBLE Transcript ENSMUST00000000631). This insertion is predicted to cause a frameshift mutation, which will result in early termination and truncation of the mGluR6 protein. This truncation occurs at amino acid 701, which is located in predicted transmembrane domain IV (Nakajima et al., Reference Nakajima, Iwakabe, Akazawa, Nawa, Shigemoto, Mizuno and Nakanishi1993). In addition, seven amino acids are added to the carboxy terminus that are encoded by the insertion and prior to a stop codon that occurs in the insertion. Given the loss of several transmembrane domains and the intracellular carboxy region, this truncation would not be functional and likely the mRNA or protein if produced would be rapidly degraded.

Fig. 2.

A splice site mutation causes the Grm6 nob7 phenotype. (A) The control and Grm6 nob7 cDNA and genomic sequences are shown aligned in the region of interest. The Grm6 nob7 cDNA (B) and genomic DNA (C) have a G to A mutation at the last nucleotide of exon 8. This is a highly conserved base at splice sites and its mutation results in the use of a cryptic splice site 28 base pairs downstream (arrows). This frameshift truncates the normal protein at amino acid 701 and the intron insertion results in the addition of seven amino acids.

BLAST comparison (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the 28 base pair sequence inserted into the Grm6 nob7 cDNA with the mouse genome database indicated that it was derived from the beginning of intron 8 of Grm6. Furthermore, the last base of exon 8, a G, was mutated to an A. This G is highly conserved at most splice donor sites because it establishes alignment with U1 snRNA during splicing. To determine if the mutation was present in the genomic DNA, we PCR-amplified fragments from genomic DNA isolated from control and Grm6 nob7 mice using primers located in exon 8 and exon 9. As shown in the partial sequence of this fragment, the last base of exon 8 contains a G to A transition (Fig. 2C). Alignment of the Grm6 nob7 cDNA with the genomic DNA shows that a cryptic splice 28 base pair downstream from the normal site at the end of exon 8 was used (Fig. 2C). We predict that this mutation, which creates a frameshift mutation truncating the normal protein by 170 amino acids, would result in a null allele.

To evaluate the expression of mGluR6 on the dendritic tips of DBCs, we used immunohistochemistry and confocal microscopy. As shown in Fig. 3, mGluR6 is localized as discrete puncta in the OPL of control retinas, which are known to represent the dendritic tips of DBCs located in the outer plexiform layer (OPL). However, this staining is absent from the Grm6 nob7 retina, confirming that the Grm6 nob7 mutation results in a null allele. Null mutations also underlie the Grm6 nob3 and Grm6 nob4 models, in which more extensive anatomical studies reveal that overall retinal and synaptic structures are preserved (Pinto et al., Reference Pinto, Vitaterna, Shimomura, Siepka, Balannik, McDearmon, Omura, Lumayag, Invergo, Glawe, Cantrell, Inayat, Olvera, Vessey, McCall, Maddox, Morgans, Young, Pletcher, Mullins, Troy and Takahashi2007; Maddox et al., Reference Maddox, Vessey, Yarbrough, Invergo, Cantrell, Inayat, Balannik, Hicks, Hawes, Byers, Smith, Hurd, Howell, Gregg, Chang, Naggert, Troy, Pinto, Nishina and McCall2008). In contrast, immunohistochemical staining using an antibody to CACNA1F, a presynaptic marker (Schmitz et al., Reference Schmitz, Königstorfer and Südhof2000), is comparable between control and Grm6 nob7 retinal sections, indicative of preserved OPL structure.

Fig. 3.

Immunohistochemistry of control (top) and Grm6 nob7 (bottom) retinas stained for mGluR6 (green) and CACNA1F (red). CACNA1F staining is comparable in both retinas and localizes to the outer plexiform layer where it opposes mGluR6 labeling in the control retina. In comparison, mGluR6 staining is not present in the Grm6 nob7 retina. Scale bar indicates 10 µm.

The availability of animal models for human retinal diseases has facilitated the evaluation of experimental therapies for many conditions (Fletcher et al., Reference Fletcher, Jobling, Vessey, Luu, Guymer and Baird2011; Farrar et al., Reference Farrar, Millington-Ward, Chadderton, Mansergh and Palfi2014), and the availability of allelic series provides resources with which to fully characterize a potential therapy. The Grm6 nob7 mutant is a new model for cCSNB and will be useful in understanding this condition and evaluating experimental therapies for the GRM6 form of cCSNB as they become available.

Acknowledgments

We are grateful to Dr. Amy Lee for providing the antibody against CACNA1F. This research was supported by grants from the U.S. National Institutes of Health (R21EY021852 to N.S.P. and R.G.G.; R01EY12354 to R.G.G.), the U.S. Veterans Administration Medical Research Service, Hope for Vision, a Foundation Fighting Blindness Center Grant to the Cole Eye Institute, Cleveland Clinic, and unrestricted awards from Research to Prevent Blindness to the Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine and to the Department of Ophthalmology & Visual Sciences, University of Louisville.

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Figure 0

Fig. 1. ERG characteristics of nob7 mice. (A) ERGs of nob7 mice lack the b-wave under dark-adapted (left) and light-adapted (right) conditions. (B) ERG waveforms of Grm6nob7 and Grm6nob3 mice have a comparable loss of the b-wave component. (C) Representative ERGs from two Grm6nob3/nob7 double heterozygotes.

Figure 1

Fig. 2. A splice site mutation causes the Grm6nob7 phenotype. (A) The control and Grm6nob7 cDNA and genomic sequences are shown aligned in the region of interest. The Grm6nob7 cDNA (B) and genomic DNA (C) have a G to A mutation at the last nucleotide of exon 8. This is a highly conserved base at splice sites and its mutation results in the use of a cryptic splice site 28 base pairs downstream (arrows). This frameshift truncates the normal protein at amino acid 701 and the intron insertion results in the addition of seven amino acids.

Figure 2

Fig. 3. Immunohistochemistry of control (top) and Grm6nob7 (bottom) retinas stained for mGluR6 (green) and CACNA1F (red). CACNA1F staining is comparable in both retinas and localizes to the outer plexiform layer where it opposes mGluR6 labeling in the control retina. In comparison, mGluR6 staining is not present in the Grm6nob7 retina. Scale bar indicates 10 µm.