Publications by authors named "Xi-Qin Ding"

53 Publications

Preservation of endoplasmic reticulum (ER) Ca stores by deletion of inositol-1,4,5-trisphosphate receptor type 1 promotes ER retrotranslocation, proteostasis, and protein outer segment localization in cyclic nucleotide-gated channel-deficient cone photoreceptors.

FASEB J 2021 Jun;35(6):e21579

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Endoplasmic reticulum (ER) Ca homeostasis relies on an appropriate balance between efflux- and influx-channel activity responding to dynamic changes of intracellular Ca levels. Dysregulation of this complex signaling network has been shown to contribute to neuronal and photoreceptor death in neuro- and retinal degenerative diseases, respectively. In mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model of achromatopsia/cone dystrophy, cones display early-onset ER stress-associated apoptosis and protein mislocalization. Cones in these mice also show reduced cytosolic Ca level and subsequent elevation in the ER Ca -efflux-channel activity, specifically the inositol-1,4,5-trisphosphate receptor type 1 (IP R1), and deletion of IP R1 results in preservation of cones. This work investigated how preservation of ER Ca stores leads to cone protection. We examined the effects of cone specific deletion of IP R1 on ER stress responses/cone death, protein localization, and ER proteostasis/ER-associated degradation. We demonstrated that deletion of IP R1 improves trafficking of cone-specific proteins M-/S-opsin and phosphodiesterase 6C to cone outer segments and reduces localization to cone inner segments. Consistent with the improved protein localization, deletion of IP R1 results in increased ER retrotranslocation protein expression, reduced proteasome subunit expression, reduced ER stress/cone death, and reduced retinal remodeling. We also observed the enhanced ER retrotranslocation in mice that have been treated with a chemical chaperone, supporting the connection between improved ER retrotranslocation/proteostasis and alleviation of ER stress. Findings from this work demonstrate the importance of ER Ca stores in ER proteostasis and protein trafficking/localization in photoreceptors, strengthen the link between dysregulation of ER Ca homeostasis and ER stress/cone degeneration, and support an involvement of improved ER proteostasis in ER Ca preservation-induced cone protection; thereby identifying IP R1 as a critical mediator of ER stress and protein mislocalization and as a potential target to preserve cones in CNG channel deficiency.
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http://dx.doi.org/10.1096/fj.202002711RDOI Listing
June 2021

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the -Deficient Mouse Model of Achromatopsia.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-University, 81377 Munich, Germany.

Mutations in the gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in KO mice. Virus-mediated knockdown or genetic ablation of in KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.
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http://dx.doi.org/10.3390/ijms22010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793084PMC
December 2020

Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling-induced cone degeneration in cyclic nucleotide-gated channel deficiency.

FASEB J 2020 05 16;34(5):6335-6350. Epub 2020 Mar 16.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling and upregulation of the ER Ca channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration.
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http://dx.doi.org/10.1096/fj.201901951RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299158PMC
May 2020

Inhibition of thyroid hormone signaling protects retinal pigment epithelium and photoreceptors from cell death in a mouse model of age-related macular degeneration.

Cell Death Dis 2020 01 13;11(1):24. Epub 2020 Jan 13.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Dry AMD is characterized by a progressive macular degeneration of the retinal pigment epithelium (RPE) and photoreceptors, and the RPE oxidative damage/dystrophy is at the core of the disease. Recent population/patients-based studies have shown an association of high free serum thyroid hormone (TH) levels with increased risk of AMD. This work investigated the effects of TH signaling inhibition on RPE and photoreceptor damage/cell death in an oxidative stress-induced mouse model of AMD. TH signaling inhibition was achieved by anti-thyroid drug treatment and oxidative stress was induced by sodium iodate (NaIO) administration. Mice treated with NaIO showed severe RPE and photoreceptor cell death/necroptosis, destruction, oxidative damage, retinal stress, and reduced retinal function. Treatment with anti-thyroid drug protected RPE and photoreceptors from damage/cell death induced by NaIO, reduced oxidative damage of RPE and photoreceptors, and preserved retinal function. Gene expression analysis showed that the NaIO-induced RPE/photoreceptor damage/cell death involves multiple mechanisms, including cellular oxidative stress responses, activation of necroptosis/apoptosis signaling, and inflammatory responses. Treatment with anti-thyroid drug abolished these cellular stress/death responses. The findings of this study demonstrate a role of TH signaling in RPE and photoreceptor cell death after oxidative stress challenge, and support a role of TH signaling in the pathogenesis of AMD.
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http://dx.doi.org/10.1038/s41419-019-2216-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957507PMC
January 2020

Ryanodine Receptor 2 Contributes to Impaired Protein Localization in Cyclic Nucleotide-Gated Channel Deficiency.

eNeuro 2019 May/Jun;6(3). Epub 2019 Jun 27.

Department of Cell Biology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma 73104

The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. Mutations in the cone photoreceptor CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone apoptosis, protein mislocalization, and ER calcium dysregulation. This work investigated the potential mechanisms of protein mislocalization associated with ER calcium dysregulation using mice lacking ER Ca channel ryanodine receptor 2 (RyR2) specifically in cones. Deletion of improved outer segment (OS) localization of the cone proteins M-opsin, S-opsin, and cone phosphodiesterase subunit α' (PDE6C) and decreased inner segment localization. One-month-old mice showed ∼30% of M-opsin, 55% of S-opsin, and 50% of PDE6C localized to the OS. mice with deletion at the same age showed almost 60% of M-opsin, 70% of S-opsin, and 70% of PDE6C localized to the OS. Deletion of nearly completely reversed elevations of the ER stress markers phospho-IRE1α and phospho-eIF2α and suppressed cone apoptosis. Consistent with the improved cone protein localization and reduced ER stress/cone apoptosis, cone survival was improved by deletion of The number of cones was increased by ∼28% in 2- to 4-month-old mice with deletion compared with age-matched mice. This work demonstrates a role of RyR2/ER calcium dysregulation in protein mislocalization, ER stress, and cone death. The findings provide novel insights into the mechanisms of photoreceptor degeneration and support strategies targeting ER calcium regulation to manage retinal degeneration.
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http://dx.doi.org/10.1523/ENEURO.0119-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597858PMC
March 2020

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

J Clin Invest 2018 12 12;128(12):5663-5675. Epub 2018 Nov 12.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
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http://dx.doi.org/10.1172/JCI96098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264655PMC
December 2018

Deficiency of type 2 iodothyronine deiodinase reduces necroptosis activity and oxidative stress responses in retinas of Leber congenital amaurosis model mice.

FASEB J 2018 Jun 6:fj201800484RR. Epub 2018 Jun 6.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

Thyroid hormone (TH) signaling has been shown to regulate cone photoreceptor viability. Suppression of TH signaling with antithyroid drug treatment or by targeting iodothyronine deiodinases and TH receptors preserves cones in mouse models of retinal degeneration, including the Leber congenital amaurosis Rpe65-deficient mice. This work investigates the cellular mechanisms underlying how suppressing TH signaling preserves cones in Rpe65-deficient mice, using mice deficient in type 2 iodothyronine deiodinase (Dio2), the enzyme that converts the prohormone thyroxine to the active hormone triiodothyronine (T3). Deficiency of Dio2 improved cone survival and function in Rpe65 and Rpe65-deficiency on a cone dominant background ( Rpe65/ Nrl) mice. Analysis of cell death pathways revealed that receptor-interacting serine/threonine-protein kinase (RIPK)/necroptosis activity was increased in Rpe65/ Nrl retinas, and Dio2 deficiency reversed the alterations. Cell-stress analysis showed that the cellular oxidative stress responses were increased in Rpe65/ Nrl retinas, and Dio2 deficiency abolished the elevations. Similarly, antithyroid drug treatment resulted in reduced RIPK/necroptosis activity and oxidative stress responses in Rpe65/ Nrl retinas. Moreover, treatment with T3 significantly induced RIPK/necroptosis activity and oxidative stress responses in the retina. This work shows that suppression of TH signaling reduces cellular RIPK/necroptosis activity and oxidative stress responses in degenerating retinas, suggesting a mechanism underlying the observed cone preservation.-Yang, F., Ma, H., Butler, M. R., Ding, X.-Q. Deficiency of type 2 iodothyronine deiodinase reduces necroptosis activity and oxidative stress responses in retinas of Leber congenital amaurosis model mice.
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http://dx.doi.org/10.1096/fj.201800484RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181634PMC
June 2018

Overexpression of Type 3 Iodothyronine Deiodinase Reduces Cone Death in the Leber Congenital Amaurosis Model Mice.

Adv Exp Med Biol 2018 ;1074:125-131

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Leber congenital amaurosis (LCA) is a devastating pediatric retinal degenerative disease, accounting for 20% of blindness in children attending schools for the blind. Mutations in the RPE65 gene, which encodes the retinal pigment epithelium-specific isomerohydrolase RPE65, account for 16% of all LCA cases. Recent findings have linked cone photoreceptor viability to thyroid hormone (TH) signaling. TH signaling regulates cell proliferation, differentiation, and metabolism. At the cellular level, TH action is regulated by the two iodothyronine deiodinases, DIO2 and DIO3. DIO2 converts the prohormone thyroxine (T4) to the bioactive hormone triiodothyronine (T3), and DIO3 inactivates T3 and T4. The present work investigates the effects of overexpression of DIO3 to suppress TH signaling and thereby modulate cone death/survival. Subretinal delivery of AAV5-IRBP/GNAT2-hDIO3 induced robust expression of DIO3 in the mouse retina and significantly reduced the number of TUNEL-positive cells in the cone-dominant LCA model Rpe65 /Nrl mice. Our work shows that suppressing TH signaling by overexpression of DIO3 preserves cones, supporting that suppressing TH signaling locally in the retina may represent a treatment strategy for LCA management.
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http://dx.doi.org/10.1007/978-3-319-75402-4_16DOI Listing
May 2019

Thyroid Hormone Signaling in Retinal Development, Survival, and Disease.

Vitam Horm 2018 19;106:333-349. Epub 2017 Jun 19.

University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States. Electronic address:

Thyroid hormone (TH) is essential in numerous physiological functions and developmental processes. It acts through TH receptors (TRs) to regulate gene expression. The retina is the light-sensitive tissue lining the back of the eye and functions as the first step of the visual process. Rod and cone photoreceptors are specialized sensory neurons in the retina that initiate phototransduction. Rods are responsible for dim light vision, whereas cones are responsible for daytime vision, color vision, and visual acuity. TH signaling regulates retinal development and maintenance. The requirement of TH signaling is typically manifested as its regulation in the cone maturation and expression of the light-sensing pigment protein (cone opsin). There are two components of this regulation. First, TRβ2, a TH-activated transcription factor, is expressed in immature cones and regulates cone differentiation and cone opsin expression; activation of TRβ2 suppresses the expression of short-wave-sensitive opsin 1, induces the expression of medium-wave-sensitive opsin 1, and promotes dorsal-ventral opsin patterning. Second, hypothyroid mouse models display abnormalities in cone opsin expression, supporting the necessity of TH itself in retinal development. TH has been linked to photoreceptor survival. Excessive TH signaling leads to death of developing photoreceptors in healthy and diseased retina, whereas suppressing TH signaling preserves cones in mouse models of retinal degeneration. Some eye diseases, including age-related macular degeneration, have been associated with elevated circulation TH levels. Future work should aim to better understand how TH regulates retinal development, functionality, and survival, to examine the role of TH signaling in the pathogenesis of retinal degeneration, and to explore the potential of TH signaling manipulation for photoreceptor protection. Hopefully, these knowledge bases will lead to the identification of novel strategies for retinal disease prevention and management.
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http://dx.doi.org/10.1016/bs.vh.2017.05.001DOI Listing
October 2018

Endoplasmic reticulum (ER) Ca-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency.

J Biol Chem 2017 07 11;292(27):11189-11205. Epub 2017 May 11.

From the Departments of Cell Biology,

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca stores through pharmacological and genetic suppression of ER Ca efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IPR1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IPR1 signaling contributes to Ca homeostasis and cone survival. Consistent with the important contribution of organellar Ca signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.
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http://dx.doi.org/10.1074/jbc.M117.782326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500788PMC
July 2017

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

FASEB J 2017 08 20;31(8):3425-3438. Epub 2017 Apr 20.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA;

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, and , encode TRs; 2 has been associated with cone viability. Using TR antagonists and deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the mouse model of Leber congenital amaurosis and reduced the number of TUNEL cells. Cone survival was significantly improved in and (a model of achromatopsia with defect) mice with deletion. Ventral cone density in and / mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.
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http://dx.doi.org/10.1096/fj.201601166RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503703PMC
August 2017

Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration.

FASEB J 2016 12 13;30(12):4313-4325. Epub 2016 Sep 13.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA;

Recent studies have implicated thyroid hormone (TH) signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we found that antithyroid treatment preserves cones. This work investigates the significance of targeting intracellular TH components locally in the retina. The cellular TH level is mainly regulated by deiodinase iodothyronine (DIO)-2 and -3. DIO2 converts thyroxine (T4) to triiodothyronine (T3), which binds to the TH receptor, whereas DIO3 degrades T3 and T4. We examined cone survival after overexpression of DIO3 and inhibition of DIO2 and demonstrated the benefits of these manipulations. Subretinal delivery of AAV5-IRBP/GNAT2-DIO3, which directs expression of human DIO3 specifically in cones, increased cone density by 30-40% in a Rpe65 mouse model of Lebers congenital amaurosis (LCA) and in a Cpfl1 mouse with Pde6c defect model of achromatopsia, compared with their respective untreated controls. Intravitreal and topical delivery of the DIO2 inhibitor iopanoic acid also significantly improved cone survival in the LCA model mice. Moreover, the expression levels of DIO2 and Slc16a2 were significantly higher in the diseased retinas, suggesting locally elevated TH signaling. We show that targeting DIOs protects cones, and intracellular inhibition of TH components locally in the retina may represent a novel strategy for retinal degeneration management.-Yang, F., Ma, H., Belcher, J., Butler, M. R., Redmond, T. M., Boye, S. L., Hauswirth, W. W., Ding, X.-Q. Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration.
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http://dx.doi.org/10.1096/fj.201600715RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102114PMC
December 2016

The B3 Subunit of the Cone Cyclic Nucleotide-gated Channel Regulates the Light Responses of Cones and Contributes to the Channel Structural Flexibility.

J Biol Chem 2016 Apr 18;291(16):8721-34. Epub 2016 Feb 18.

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, Saint Louis, Missouri 63110.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3(-/-)/Nrl(-/-) mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3(-/-)/Nrl(-/-) mice compared with Nrl(-/-) mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors.
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http://dx.doi.org/10.1074/jbc.M115.696138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861441PMC
April 2016

Thyroid Hormone Signaling and Cone Photoreceptor Viability.

Adv Exp Med Biol 2016 ;854:613-8

The Department of Cell Biology, University of Oklahoma Health Sciences Center, 73104, Oklahoma City, OK, USA.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and apoptosis. In the retina, TH signaling plays a central role in cone opsin expression. TH signaling inhibits S opsin expression, stimulates M opsin expression, and promotes dorsal-ventral opsin patterning. TH signaling has also been associated with cone photoreceptor viability. Treatment with thyroid hormone triiodothyronine (T3) or induction of high T3 by deleting the hormone-inactivating enzyme type 3 iodothyronine deiodinase (DIO3) causes cone death in mice. This effect is reversed by deletion of the TH receptor (TR) gene. Consistent with the T3 treatment effect, suppressing TH signaling preserves cones in mouse models of retinal degeneration. The regulation of cone survival by TH signaling appears to be independent of its regulatory role in cone opsin expression. The mechanism by which TH signaling regulates cone viability remains to be identified. The current understanding of TH signaling regulation in photoreceptor viability suggests that suppressing TH signaling locally in the retina may represent a novel strategy for retinal degeneration management.
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http://dx.doi.org/10.1007/978-3-319-17121-0_81DOI Listing
April 2016

Gene Therapy Fully Restores Vision to the All-Cone Nrl(-/-) Gucy2e(-/-) Mouse Model of Leber Congenital Amaurosis-1.

Hum Gene Ther 2015 Sep 6;26(9):575-92. Epub 2015 Aug 6.

1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.

Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.
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http://dx.doi.org/10.1089/hum.2015.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575531PMC
September 2015

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice.

J Biol Chem 2015 Aug 29;290(34):20880-20892. Epub 2015 Jun 29.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104. Electronic address:

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.
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http://dx.doi.org/10.1074/jbc.M115.641159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543649PMC
August 2015

Vitreal delivery of AAV vectored Cnga3 restores cone function in CNGA3-/-/Nrl-/- mice, an all-cone model of CNGA3 achromatopsia.

Hum Mol Genet 2015 Jul 8;24(13):3699-707. Epub 2015 Apr 8.

Deparment of Ophthalmology, University of Florida, Gainesville, FL 32610, USA, School of Ophthalmology and Optometry, The Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China, Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University and State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China,

The CNGA3(-/-)/Nrl(-/-) mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function-structure could be rescued in CNGA3(-/-)/Nrl(-/-) mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function.
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http://dx.doi.org/10.1093/hmg/ddv114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459390PMC
July 2015

Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

Adv Exp Med Biol 2014 ;801:57-65

Departments of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 553, 73104, Oklahoma City, OK, USA,

Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.
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http://dx.doi.org/10.1007/978-1-4614-3209-8_8DOI Listing
July 2014

Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration.

Proc Natl Acad Sci U S A 2014 Mar 18;111(9):3602-7. Epub 2014 Feb 18.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104.

Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling, which regulates cell proliferation, differentiation, and apoptosis, plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models. Retinol isomerase RPE65-deficient mice [a model of Leber congenital amaurosis (LCA) with rapid cone loss] and cone photoreceptor function loss type 1 mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone death. Further, cone cyclic nucleotide-gated channel B subunit-deficient mice (moderate achromatopsia) and guanylate cyclase 2e-deficient mice (LCA with slower cone loss) were used to determine whether triiodothyronine (T3) treatment (stimulating TH signaling) causes deterioration of cones. We found that cone density in retinol isomerase RPE65-deficient and cone photoreceptor function loss type 1 mice increased about sixfold following antithyroid treatment. Cone density in cone cyclic nucleotide-gated channel B subunit-deficient and guanylate cyclase 2e-deficient mice decreased about 40% following T3 treatment. The effect of TH signaling on cone viability appears to be independent of its regulation on cone opsin expression. This work demonstrates that suppressing TH signaling in retina dystrophy mouse models is protective of cones, providing insights into cone preservation and therapeutic interventions.
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http://dx.doi.org/10.1073/pnas.1317041111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948228PMC
March 2014

cGMP accumulation causes photoreceptor degeneration in CNG channel deficiency: evidence of cGMP cytotoxicity independently of enhanced CNG channel function.

J Neurosci 2013 Sep;33(37):14939-48

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Center for Integrated Protein Science Munich and Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, 80539 Munich, Germany, John A. Moran Eye Center, University of Utah, Salt Lake City, Utah 84132, and Department of Basic Sciences and Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.
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http://dx.doi.org/10.1523/JNEUROSCI.0909-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771030PMC
September 2013

Loss of cone cyclic nucleotide-gated channel leads to alterations in light response modulating system and cellular stress response pathways: a gene expression profiling study.

Hum Mol Genet 2013 Oct 4;22(19):3906-19. Epub 2013 Jun 4.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophy. We investigated the gene expression profiles in mouse retina with CNG channel deficiency using whole genome expression microarrays. As cones comprise only 2 to 3% of the total photoreceptor population in the wild-type mouse retina, the mouse lines with CNG channel deficiency on a cone-dominant background, i.e. Cnga3-/-/Nrl-/- and Cngb3-/-/Nrl-/- mice, were used in our study. Comparative data analysis revealed a total of 105 genes altered in Cnga3-/-/Nrl-/- and 92 in Cngb3-/-/Nrl-/- retinas, relative to Nrl-/- retinas, with 27 genes changed in both genotypes. The differentially expressed genes primarily encode proteins associated with cell signaling, cellular function maintenance and gene expression. Ingenuity pathway analysis (IPA) identified 26 and 9 canonical pathways in Cnga3-/-/Nrl-/- and Cngb3-/-/Nrl-/- retinas, respectively, with 6 pathways being shared. The shared pathways include phototransduction, cAMP/PKA-mediated signaling, endothelin signaling, and EIF2/endoplasmic reticulum (ER) stress, whereas the IL-1, CREB, and purine metabolism signaling were found to specifically associate with Cnga3 deficiency. Thus, CNG channel deficiency differentially regulates genes that affect cell processes such as phototransduction, cellular survival and gene expression, and such regulations play a crucial role(s) in the retinal adaptation to impaired cone phototransduction. Though lack of Cnga3 and Cngb3 shares many common pathways, deficiency of Cnga3 causes more significant alterations in gene expression. This work provides insights into how cones respond to impaired phototransduction at the gene expression levels.
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http://dx.doi.org/10.1093/hmg/ddt245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766184PMC
October 2013

Detection of cGMP in the degenerating retina.

Methods Mol Biol 2013 ;1020:235-45

Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität, Munich, Germany.

Cyclic guanosine 3'-5'-monophosphate (cGMP) plays a key role in the physiological process of light detection in photoreceptor cells of the retina. However, there is also growing evidence that cGMP may be critically involved in some pathophysiological processes of the retina since degenerating photoreceptors in mouse models of retinitis pigmentosa and achromatopsia accumulate high levels of cGMP. Here, we describe methods that allow the detection, subcellular localization, and quantification of cGMP in the retina and propose that cGMP accumulation can be used as a biomarker for photoreceptor degeneration.
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http://dx.doi.org/10.1007/978-1-62703-459-3_16DOI Listing
December 2013

Cones respond to light in the absence of transducin β subunit.

J Neurosci 2013 Mar;33(12):5182-94

Departments of Neuroscience and Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA.

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gβ3 and Gγt2 subunits. The function of Gβγ in this cascade has not been examined. Here, we investigate the role of Gβ3 by assessing cone structure and function in Gβ3-null mouse (Gnb3(-/-)). We found that Gβ3 is required for the normal expression of its partners, because in the Gnb3(-/-) cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3(-/-) cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gβ subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3(-/-) cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gβ3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.
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http://dx.doi.org/10.1523/JNEUROSCI.5204-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866503PMC
March 2013

Endoplasmic reticulum stress-associated cone photoreceptor degeneration in cyclic nucleotide-gated channel deficiency.

J Biol Chem 2012 May 9;287(22):18018-29. Epub 2012 Apr 9.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.

Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for >70% of all known cases of achromatopsia. Cones degenerate in achromatopsia patients and in CNGA3(-/-) and CNGB3(-/-) mice. This work investigates the molecular basis of cone degeneration in CNG channel deficiency. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we generated mouse lines with CNG channel deficiency on a cone-dominant background, i.e. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice. The retinal phenotype and potential cell death pathways were examined by functional, biochemical, and immunohistochemical approaches. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice showed impaired cone function, opsin mislocalization, and cone degeneration similar to that in the single knock-out mice. The endoplasmic reticulum stress marker proteins, including Grp78/Bip, phospho-eIF2α, phospho-IP(3)R, and CCAAT/enhancer-binding protein homologous protein, were elevated significantly in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, compared with the age-matched (postnatal 30 days) Nrl(-/-) controls. Along with these, up-regulation of the cysteine protease calpains and cleavage of caspase-12 and caspase-7 were found in the channel-deficient retinas, suggesting an endoplasmic reticulum stress-associated apoptosis. In addition, we observed a nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, implying a mitochondrial insult in the endoplasmic reticulum stress-activated cell death process. Taken together, our findings suggest a crucial role of endoplasmic reticulum stress in cone degeneration associated with CNG channel deficiency.
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http://dx.doi.org/10.1074/jbc.M112.342220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365688PMC
May 2012

CNGA3 deficiency affects cone synaptic terminal structure and function and leads to secondary rod dysfunction and degeneration.

Invest Ophthalmol Vis Sci 2012 Mar 1;53(3):1117-29. Epub 2012 Mar 1.

Department of Cell Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

Purpose: To investigate rod function and survival after cone dysfunction and degeneration in a mouse model of cone cyclic nucleotide-gated (CNG) channel deficiency.

Methods: Rod function and survival in mice with cone CNG channel subunit CNGA3 deficiency (CNGA3-/- mice) were evaluated by electroretinographic (ERG), morphometric, and Western blot analyses. The arrangement, integrity, and ultrastructure of photoreceptor terminals were investigated by immunohistochemistry and electron microscopy.

Results: The authors found loss of cone function and cone death accompanied by impairment of rods and rod-driven signaling in CNGA3-/- mice. Scotopic ERG b-wave amplitudes were reduced by 15% at 1 month, 30% at 6 months, and 40% at 9 months and older, while scotopic a-wave amplitudes were decreased by 20% at 9 months, compared with ERGs of age-matched wild-type mice. Outer nuclear layer thickness in CNGA3-/- retina was reduced by 15% at 12 months compared with age-matched wild-type controls. This was accompanied by a 30%-40% reduction in expression of rod-specific proteins, including rhodopsin, rod transducin α-subunit, and glutamic acid-rich protein (GARP). Cone terminals in the CNGA3-/- retina showed a progressive loss of neurochemical and ultrastructural integrity. Abnormalities were observed as early as 1 month. Disorganized rod terminal ultrastructure was noted by 12 months.

Conclusions: These findings demonstrate secondary rod impairment and degeneration after cone degeneration in mice with cone CNG channel deficiency. Loss of cone phototransduction accompanies the compromised integrity of cone terminals. With time, rod synaptic structure, function, and viability also become compromised.
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http://dx.doi.org/10.1167/iovs.11-8168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339899PMC
March 2012

Overexpression of ROM-1 in the cone-dominant retina.

Adv Exp Med Biol 2012 ;723:633-9

Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, BMS 781, Oklahoma City, OK 73104, USA.

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http://dx.doi.org/10.1007/978-1-4614-0631-0_80DOI Listing
April 2012

Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy.

Hum Mol Genet 2011 Aug 15;20(16):3161-75. Epub 2011 May 15.

The Department of Genetics, UCL Institute of Ophthalmology, London, UK.

Mutations in the CNGB3 gene account for >50% of all known cases of achromatopsia. Although of early onset, its stationary character and the potential for rapid assessment of restoration of retinal function following therapy renders achromatopsia a very attractive candidate for gene therapy. Here we tested the efficacy of an rAAV2/8 vector containing a human cone arrestin promoter and a human CNGB3 cDNA in CNGB3 deficient mice. Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins. Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity. Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old). This study represents achievement of the most substantial restoration of visual function reported to date in an animal model of achromatopsia using a human gene construct, which has the potential to be utilized in clinical trials.
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http://dx.doi.org/10.1093/hmg/ddr218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3140821PMC
August 2011

Early-onset, slow progression of cone photoreceptor dysfunction and degeneration in CNG channel subunit CNGB3 deficiency.

Invest Ophthalmol Vis Sci 2011 Jun 1;52(6):3557-66. Epub 2011 Jun 1.

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

Purpose: To investigate the progression of cone dysfunction and degeneration in CNG channel subunit CNGB3 deficiency.

Methods: Retinal structure and function in CNGB3(-/-) and wild-type (WT) mice were evaluated by electroretinography (ERG), lectin cytochemistry, and correlative Western blot analysis of cone-specific proteins. Cone and rod terminal integrity was assessed by electron microscopy and synaptic protein immunohistochemical distribution.

Results: Cone ERG amplitudes (photopic b-wave) in CNGB3(-/-) mice were reduced to approximately 50% of WT levels by postnatal day 15, decreasing further to approximately 30% of WT levels by 1 month and to approximately 20% by 12 months of age. Rod ERG responses (scotopic a-wave) were not affected in CNGB3(-/-) mice. Average CNGB3(-/-) cone densities were approximately 80% of WT levels at 1 month and declined slowly thereafter to only approximately 50% of WT levels by 12 months. Expression levels of M-opsin, cone transducin α-subunit, and cone arrestin in CNGB3(-/-) mice were reduced by 50% to 60% by 1 month and declined to 35% to 45% of WT levels by 9 months. In addition, cone opsin mislocalized to the outer nuclear layer and the outer plexiform layer in the CNGB3(-/-) retina. Cone and rod synaptic marker expression and terminal ultrastructure were normal in the CNGB3(-/-) retina.

Conclusions: These findings are consistent with an early-onset, slow progression of cone functional defects and cone loss in CNGB3(-/-) mice, with the cone signaling deficits arising from disrupted phototransduction and cone loss rather than from synaptic defects.
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http://dx.doi.org/10.1167/iovs.10-6358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109041PMC
June 2011

Molecular pathogenesis of achromatopsia associated with mutations in the cone cyclic nucleotide-gated channel CNGA3 subunit.

Adv Exp Med Biol 2010 ;664:245-53

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

Cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are linked to achromatopsia and progressive cone dystrophy in humans. Over 50 mutations have been identified in the CNGA3 subunit. The R277C and R283W substitutions are among the most frequently occurring mutations. This study investigated the defects of these two mutations using a heterologous expression system. The wild type and mutant CNGA3 were expressed in HEK293 cells, the channel's expression and cellular localization were examined by immunoblotting and immunofluorecences labeling, and activity of the channel was evaluated by ratiometric [Ca(2+)](i) measurements and by electrophysiological recordings. By using this model system we observed dysfunction of the mutant channels. Co-expression of the mutant channel with the wild type subunit did not affect the wild type channel's activity. Immunoflurescence labeling showed apparent cytosol aggregation of the immunoreactivity in cells expressing the mutants. Thus these disease-causing mutations appear to induce loss of function by impairing the channel cellular trafficking and plasma membrane targeting. Therapeutic supplementation of the wild type transgene may help correct the visual disorders caused by these two mutations.
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http://dx.doi.org/10.1007/978-1-4419-1399-9_28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370940PMC
September 2011