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Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice

Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice

  • Christian Schön, 
  • François Paquet-Durand, 
  • Stylianos Michalakis
PLOS
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Abstract

Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+—signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG) channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC). Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

Introduction

Retinitis pigmentosa (RP) is characterized by a primary loss of rod photoreceptor function and structure. Subsequent non-cell autonomous degenerative processes eventually affect cone function and structure resulting in compromised daylight vision and in severe cases legal blindness.

The most extensively studied RP model is the rd1 mutant mouse [1]—a naturally occurring strain expressing a loss-of-function mutation in the Pde6b gene encoding the β-subunit of the rod-specific cGMP phosphodiesterase 6 [2]. This mutation results in a fast progressive degeneration of rod photoreceptors during postnatal development of the retina. During pathogenesis, several important processes, including non-apoptotic, cell-intrinsic factors, trigger photoreceptor death [3]. One known key player is the elevated level of intracellular Ca2+ in degenerating photoreceptors of rd1 mutant mice [4]. As a consequence, increased activity levels of the Ca2+—dependent protease calpain were found in different models of photoreceptor degeneration [3]. Ca2+ and calpain are therefore considered as potential executers of photoreceptor cell death. Currently, two different types of ion channels are known to mediate Ca2+—influx into photoreceptors: the cyclic nucleotide-gated (CNG) channel found in the outer segment plasma membrane [5] and the synaptic L-type voltage-gated Ca2+ channel (VGCC) [6, 7]. Given the importance of Ca2+—influx for photoreceptor degeneration [8], the Ca2+-conducting CNG channels and VGCCs were proposed as potential therapeutic targets for RP [4, 9]. However, pharmacological attempts to block Ca2+ influx have led to contradicting findings [813]. Genetic studies helped clarifying this discrepancy and showed that the knockout of the Cngb1 gene encoding for the β1-subunit of the rod CNG channel significantly delayed photoreceptor degeneration in rd1mt/mt mice [14, 15]. Genetic deletion of Cacnb2 encoding the β2-subunit of the VGCC also resulted in a significant, but only transient decrease in the rate of photoreceptor degeneration in rd1mt/mt mice (15).

In photoreceptors, the β2-subunit was suggested as the preferred β-subunit associated with Cav1.4 channels [16, 17]. However, the pore-forming Cav1.4 α1 subunit can in principle also functionally associate with other β-subunits [18, 19] which might also exist in the retina.

To define the role of the pore-forming Cav1.4 α1 subunit of the VGCC channel on photoreceptor degeneration, we cross-bred the rd1 model with Cacna1f-deficient mice [20]. We found that genetic deletion of Cacna1f led to a significant, but only short-term delay of photoreceptor degeneration in rd1 mice. However, knockout of Cacna1f prevented excess activation of calpain commonly seen in degenerating photoreceptors (3), highlighting the dependence of this phenomenon on synaptic VGCCs.

Material and Methods

Ethics statement

The mouse studies were approved by the local authority (Regierung von Oberbayern, Az. 55.2-1-54-2532-50-15) and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anesthetized by intraperitoneal injections of ketamine (0.05 mg/g) and xylazine (0.01 mg/g) and were sacrificed by cervical dislocation. All efforts were taken to minimize suffering of the animals.

Animals

In this study, we compared Pde6brd1 mutant mice (rd1mt/mt) with double mutant mice carrying the homozygous rd1 mutation and lacking expression of the X-chromosomal Cacna1f gene (rd1mt/mt x Cacna1fko/ko). Cacna1fko/ko mice were provided by Dr Marion Maw, University of Otago, Dunedin, New Zealand [20]. Due to the lack of the pore-forming Cav1.4 α1 subunit of the VGCC these mice develop a rod and cone photoreceptor synaptopathy and cone but not rod photoreceptor degeneration [21, 22].

Optical coherence tomography (OCT) measurements

Before the scanning procedure, mice were anesthetized and Tropicamide eye drops were applied to the animals’ eyes for pupil dilation (Mydriadicum Stulln, Pharma Stulln GmbH, Stulln, Germany). Subsequently, hydroxylpropyl methylcellulose (Methocel 2%; OmniVision, Puchheim, Germany) was applied to keep the eyes moist. The OCT examinations were performed with a MICRON IV system (Phoenix research labs, Pleasanton, United States). A vertical OCT scan was centered to the optic nerve head and measurements were conducted in the dorsal part of the retina. InSight software (Phoenix research labs) was used to measure the photoreceptor layer thickness as the distance between the outer plexiform layer and the border of neuronal retina and the retinal pigment epithelium. The photoreceptor layer thickness as defined in our study is also known as “photoreceptor plus” or REC + [23].

Calpain activity assay

To test for calpain activity an enzymatic in situ assay was used [24]. Unfixed retinal cryosections were pre-incubated for 15 min in calpain reaction buffer (CRB; 25 mM HEPES, 65 mM KCl, 2 mM MgCl2, 1,5 mM CaCl2, 2 mM DTT) and then incubated at 35°C for 1 hour in the dark in CRB with 2 mM fluorescent calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-Leucyl-L-methionine amide (CMAC, t-BOC-Leu-Met; Molecular Probes, Eugene, USA). Calpain-dependent cleavage of the substrate led to the uncaging of fluorescence, which was then used for quantifying the numbers of calpain activity positive cells.

TUNEL assay

The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was performed using an in situ cell death detection kit (Fluorescein or TMR; Roche Diagnostics GmbH, Mannheim, Germany). As negative control the terminal deoxynucleotidyl transferase enzyme was omitted from the labelling solution, for positive control the sections were pre-treated for 30 min with DNAse I (Roche, 3 U/ml) in 50 mM Tris-HCl, pH 7.5, 1 mg/ml BSA to induce DNA strand breaks. Negative control gave no staining, while positive control stained all nuclei in all layers of the retina [25].

Microscopy and cell counting

Fluorescence microscopy was performed on a Z1 ApoTome Microscope equipped with a Zeiss Axiocam digital camera (Zeiss, Oberkochen, Germany). Images were captured using Zeiss Axiovision 4.7 software and representative pictures were taken from central areas of the retina. Adobe Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA) was used for image processing and figure assemblies.

For cell quantification, pictures were captured on whole radial sections using the Mosaix mode in Axiovision 4.7. Labelled cells (TUNEL, calpain activity) were counted manually. The total number of cells was determined by dividing the area of the outer nuclear layer (ONL) through the average ONL cell size. The number of positive cells was then divided by the total number of ONL cells to give the percentage of positive cells.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 5 software. To compare groups at one time-point, either Welsh's t-test or one-way ANOVA with Tukey post-test was applied. For comparing groups in longitudinal OCT examinations, two-way ANOVA with Bonferroni post-tests were performed. All values are given as mean ± SEM and n indicates the number of eyes.

Results

Cacna1f knockout delays photoreceptor cell death in rd1mt/mt mice after eye opening

To determine the influence of the synaptic VGCC on photoreceptor degeneration, we crossbred rd1mt/mt with Cacna1fko/ko animals to obtain rd1mt/mt x Cacna1fko/ko double mutant mice. Repeated OCT measurements of the retina were used to determine the photoreceptor layer thickness (receptor plus, REC+, see Methods) (Fig 1A). Right after eye opening (p13), rd1mt/mt and rd1mt/mt x Cacna1fko/ko mice already showed a severely reduced photoreceptor layer thickness in comparison to wild type animals (Fig 1A). When comparing the two models, we found no significant difference in the photoreceptor layer thickness between rd1mt/mt and rd1mt/mt x Cacna1fko/ko mice at this stage. However, at later time points, the photoreceptor layer in rd1mt/mt x Cacna1fko/ko was significantly better preserved compared to rd1mt/mt mice (p < 0.001 at p15 and p < 0.01 at p17, two-way ANOVA, n = 8), (Fig 1A and 1B). This finding was corroborated by comparing the photoreceptor rows in vertical retinal cryo-sections stained with the nuclear dye DAPI (Fig 2). Quantification at p15 revealed significantly less photoreceptor rows in rd1mt/mt (3.0 ± 0.4) compared to rd1mt/mt x Cacna1fko/ko (4.2 ± 0.2) retinas (p < 0.05, Welch's t-test, n = 4–7) confirming the in vivo OCT imaging findings.

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Fig 1. Knockout of Cacna1f delays photoreceptor cell loss in rd1mt/mt mutant mice.

(A) Representative in vivo OCT images of wild type, rd1mt/mt and rd1mt/mt x Cacna1fko/ko retinas at p15 and p17. The vertical bars mark the corresponding outer nuclear layer (ONL) thickness. (B) Quantification of photoreceptor layer thickness (REC+) of the different genotypes at p13, p15, p17, and p20. Each data point represents the mean photoreceptor layer thickness (n = 8 per genotype and time point) ± SEM; **p < 0.01, ***p < 0.001.

https://doi.org/10.1371/journal.pone.0156974.g001

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Fig 2. Knockout of Cacna1f reduces the number of dying photoreceptors in rd1mt/mt mice.

(A-C) Representative vertical cryo-sections of (A) wild type, (B) rd1mt/mt and (C) rd1mt/mt x Cacna1fko/ko retinas at p11, p13, p15, and p18 stained with TUNEL (red) and DAPI (grey). The horizontal bars mark the outer nuclear layer (ONL) borders. (D) Summary graph showing the quantification of TUNEL for the different genotypes at p11, p13, p15, and 18. Each data point represents the mean percentage of TUNEL positive cells in the ONL (n = 3–8 per genotype and time point) ± SEM; ***p < 0.0001.

https://doi.org/10.1371/journal.pone.0156974.g002

Cacna1f knockout decreases the number of dying rd1mt/mt photoreceptors before eye opening

Next, we applied TUNEL assays on vertical retinal cryo-sections to determine the extent of cell death in wild type, rd1mt/mt, and rd1mt/mt x Cacna1fko/ko mice. In comparison to the OCT data (Fig 1), the TUNEL assay provided important additional insights to the cell death kinetics before eye opening. At p11, both rd1mt/mt and rd1mt/mt x Cacna1fko/ko mice had a similar number of photoreceptor rows in comparison to wild type mice (Fig 2A–2C). At this time, both single and double mutant retinas displayed high numbers of TUNEL positive photoreceptor nuclei. However, when comparing the two models, rd1mt/mt mice showed significantly more TUNEL positive photoreceptors than rd1mt/mt x Cacna1fko/ko mice (p < 0.0001, ANOVA, n = 4–8), (Fig 2A–2D). At later time points (p13, p15, and p18) no significant difference in TUNEL positive photoreceptors could be observed. Taken together, both the in vivo OCT and the ex vivo TUNEL data suggested a significant, but not sustained delay of photoreceptor degeneration in the double mutant animals.

Calpain activation in degenerating photoreceptors depends on Cacna1f

A hallmark of photoreceptor degeneration in rd1mt/mt mice is the pronounced activation of the Ca2+ dependent protease calpain [26]. As described previously, calpain activation at least partially depends on Ca2+ entry through CNG channels in rod outer segments, since it is clearly reduced in rd1mt/mt photoreceptors lacking the CNGB1 subunit of the rod CNG channel [14]. To test if Cav1.4 channels also played a role in the activation of calpain in degenerating rd1mt/mt photoreceptors, we analyzed calpain activity in our double mutant mice. When comparing rd1mt/mt x Cacna1fko/ko mice with rd1mt/mt mice, we found an almost complete lack of calpain activation in the absence of Cav1.4 channels (Fig 3). At p11 the levels of calpain activity in double mutants were similar to wild type, while there was a significant difference to the enhanced calpain levels of rd1mt/mt mice (p < 0.0001, ANOVA, n = 4–8), (Fig 3A–3D). At later time points (p13 –p18), calpain activity increased marginally in rd1mt/mt x Cacna1fko/ko mice but never reached rd1mt/mt levels (p < 0.0001 at p13 and p15, p < 0.05 at p18, ANOVA, n = 3–8).

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Fig 3. Knockout of Cacna1f significantly prevented excess activation of calpain in rd1mt/mt photoreceptors.

(A-C) Representative vertical retinal cryo-sections of (A) wild type, (B) rd1mt/mt and (C) rd1mt/mt x Cacna1fko/ko retinas at p11, p13, p15, and p18. Photoreceptor nuclei with increased calpain activity appear as grey dots or circles. The horizontal bars mark the outer nuclear layer (ONL) borders. (D) Summary graph showing the quantification of calpain-positive photoreceptors in the different genotypes at p11, p13, p15, and 18. Each data point represents the mean percentage of cells with increased calpain activity in the ONL (n = 3–8 per genotype and time point) ± SEM; ***p < 0.0001, *p < 0.05.

https://doi.org/10.1371/journal.pone.0156974.g003

Discussion

Progressive rod and cone degeneration leads to functional blindness in RP patients. While the disease is still untreatable, various strategies emerge aiming at restoring the function or delaying the degeneration of photoreceptors. In order to provide a long-term beneficial effect a successful treatment has to achieve both, restoration of function and rescue of degeneration of rod photoreceptors.

One treatment strategy aims at decreasing intracellular Ca2+ levels to stop photoreceptor degeneration. Attempts to pharmacologically rescue photoreceptors in RP animal models by diltiazem have led to positive but contradicting findings [813]. Additionally, the Ca2+ channel blocker nilvadipine was tested in a clinical trial with RP patients resulting in promising but ambiguous effects on central visual field defects [27, 28].

In the present study we examined the role of the Cav1.4 α1 subunit of the L-type VGCC by crossing Cacna1f-deficient mice with rd1mt/mt mice. In addition to histological methods, we examined the mouse retina using in vivo OCT. This non-invasive imaging technique allowed for longitudinal examinations of the photoreceptor layer thickness in individual animals minimizing inter-individual variability. Our data on the deletion of the principal pore-forming Cav1.4 α1 subunit of the L-type VGCC in rd1mt/mt mice strengthen the view that synaptic Ca2+ entry through Cav1.4 L-type VGCC contributes to rod photoreceptor degeneration. However, together with previous findings on the analysis of rd1mt/mt x Cancnb2ko/ko mice (15) the data show that genetic deletion of the photoreceptor L-type VGCC was not able to fully rescue rod photoreceptors from degeneration and resulted only in a short-term delay of degeneration.

Interestingly, our experiments uncovered a novel major role of L-type VGCCs on calpain activation during photoreceptor degeneration. Excessive activation of calpain-type proteases can be detected in a variety of different retinal degeneration models and was previously thought to be triggered by Ca2+ influx via CNG channels (3). Our data indicates that the synaptic VGCCs are at least equally important for calpain activation. In particular, VGCCs appear to be crucial for full activation of calpain-type proteases in rd1 photoreceptors, providing evidence for a major role of synaptic Ca2+ entry in the excessive calpain activation commonly observed in degenerating photoreceptors.

The strong effect of Cacna1f-deletion on calpain activation in the rd1mt/mt retina together with the temporally restricted protection of photoreceptors suggests that other L-type VGCC- and calpain-independent pathways dictate the long-term outcome in rapid photoreceptor degeneration. In addition, contribution of structural effects caused by the loss of L-type VGCC [21, 22, 29, 30] cannot be ruled out. Remarkably, when we compare our new data with the results of our previous study on the analysis of rd1mt/mt mice lacking the CNGB1 subunit of the rod CNG channel [14], it is evident that CNG channel deletion has a relatively much stronger effect on rd1 photoreceptor survival.

In light of these results and previous findings on the neuroprotective potential of Ca2+ channel inhibitors like diltiazem and dihydropyridines it seems preferable to simultaneously target multiple channels and signaling pathways to generate synergistic effects and hopefully provide enhanced and more sustained protection of photoreceptors in retinal degeneration.

Acknowledgments

We thank Klaudija Masarini for excellent technical help and Dr. Marion Maw (University of Otago, Dunedin, New Zealand) for providing the Cacna1f-deficient mouse line.

Author Contributions

Conceived and designed the experiments: CS FPD SM. Performed the experiments: CS FPD SM. Analyzed the data: CS FPD SM. Contributed reagents/materials/analysis tools: CS FPD SM. Wrote the paper: CS FPD SM.

References

  1. 1. Keeler CE. The Inheritance of a Retinal Abnormality in White Mice. Proc Natl Acad Sci U S A. 1924;10(7):329–33. pmid:16576828; PubMed Central PMCID: PMCPMC1085675.
  2. 2. Bowes C, Li T, Danciger M, Baxter LC, Applebury ML, Farber DB. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature. 1990;347(6294):677–80. pmid:1977087.
  3. 3. Arango-Gonzalez B, Trifunovic D, Sahaboglu A, Kranz K, Michalakis S, Farinelli P, et al. Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration. PLoS One. 2014;9(11):e112142. pmid:25392995; PubMed Central PMCID: PMCPMC4230983.
  4. 4. Fox DA, Poblenz AT, He L. Calcium overload triggers rod photoreceptor apoptotic cell death in chemical-induced and inherited retinal degenerations. Ann N Y Acad Sci. 1999;893:282–5. pmid:10672249.
  5. 5. Biel M, Michalakis S. Function and dysfunction of CNG channels: insights from channelopathies and mouse models. Mol Neurobiol. 2007;35(3):266–77. pmid:17917115.
  6. 6. Mercer AJ, Thoreson WB. The dynamic architecture of photoreceptor ribbon synapses: cytoskeletal, extracellular matrix, and intramembrane proteins. Vis Neurosci. 2011;28(6):453–71. pmid:22192503; PubMed Central PMCID: PMCPMC3437624.
  7. 7. Striessnig J, Pinggera A, Kaur G, Bock G, Tuluc P. L-type Ca channels in heart and brain. Wiley Interdiscip Rev Membr Transp Signal. 2014;3(2):15–38. pmid:24683526; PubMed Central PMCID: PMCPMC3968275.
  8. 8. Barabas P, Cutler Peck C, Krizaj D. Do calcium channel blockers rescue dying photoreceptors in the Pde6b (rd1) mouse? Adv Exp Med Biol. 2010;664:491–9. pmid:20238051; PubMed Central PMCID: PMCPMC2921874.
  9. 9. Frasson M, Sahel JA, Fabre M, Simonutti M, Dreyfus H, Picaud S. Retinitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the rd mouse. Nat Med. 1999;5(10):1183–7. pmid:10502823.
  10. 10. Bush RA, Kononen L, Machida S, Sieving PA. The effect of calcium channel blocker diltiazem on photoreceptor degeneration in the rhodopsin Pro213His rat. Invest Ophthalmol Vis Sci. 2000;41(9):2697–701. pmid:10937585.
  11. 11. Pawlyk BS, Li T, Scimeca MS, Sandberg MA, Berson EL. Absence of photoreceptor rescue with D-cis-diltiazem in the rd mouse. Invest Ophthalmol Vis Sci. 2002;43(6):1912–5. pmid:12036998.
  12. 12. Pearce-Kelling SE, Aleman TS, Nickle A, Laties AM, Aguirre GD, Jacobson SG, et al. Calcium channel blocker D-cis-diltiazem does not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis pigmentosa. Mol Vis. 2001;7:42–7. pmid:11239245.
  13. 13. Sanges D, Comitato A, Tammaro R, Marigo V. Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors. Proc Natl Acad Sci U S A. 2006;103(46):17366–71. pmid:17088543; PubMed Central PMCID: PMCPMC1859935.
  14. 14. Paquet-Durand F, Beck S, Michalakis S, Goldmann T, Huber G, Muhlfriedel R, et al. A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa. Hum Mol Genet. 2011;20(5):941–7. pmid:21149284.
  15. 15. Read DS, McCall MA, Gregg RG. Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice. Exp Eye Res. 2002;75(4):415–20. pmid:12387789.
  16. 16. Ball SL, Powers PA, Shin HS, Morgans CW, Peachey NS, Gregg RG. Role of the beta(2) subunit of voltage-dependent calcium channels in the retinal outer plexiform layer. Invest Ophthalmol Vis Sci. 2002;43(5):1595–603. pmid:11980879.
  17. 17. Katiyar R, Weissgerber P, Roth E, Dorr J, Sothilingam V, Garcia Garrido M, et al. Influence of the beta2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses. Invest Ophthalmol Vis Sci. 2015;56(4):2312–24. pmid:25766584.
  18. 18. Baumann L, Gerstner A, Zong X, Biel M, Wahl-Schott C. Functional characterization of the L-type Ca2+ channel Cav1.4alpha1 from mouse retina. Invest Ophthalmol Vis Sci. 2004;45(2):708–13. pmid:14744918.
  19. 19. Koschak A, Reimer D, Walter D, Hoda JC, Heinzle T, Grabner M, et al. Cav1.4alpha1 subunits can form slowly inactivating dihydropyridine-sensitive L-type Ca2+ channels lacking Ca2+-dependent inactivation. J Neurosci. 2003;23(14):6041–9. pmid:12853422.
  20. 20. Specht D, Wu SB, Turner P, Dearden P, Koentgen F, Wolfrum U, et al. Effects of presynaptic mutations on a postsynaptic Cacna1s calcium channel colocalized with mGluR6 at mouse photoreceptor ribbon synapses. Invest Ophthalmol Vis Sci. 2009;50(2):505–15. pmid:18952919.
  21. 21. Michalakis S, Shaltiel L, Sothilingam V, Koch S, Schludi V, Krause S, et al. Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2. Hum Mol Genet. 2014;23(6):1538–50. pmid:24163243; PubMed Central PMCID: PMCPMC3929091.
  22. 22. Regus-Leidig H, Atorf J, Feigenspan A, Kremers J, Maw MA, Brandstatter JH. Photoreceptor degeneration in two mouse models for congenital stationary night blindness type 2. PLoS One. 2014;9(1):e86769. pmid:24466230; PubMed Central PMCID: PMCPMC3897778.
  23. 23. Schön C, Asteriti S, Koch S, Sothilingam V, Garcia Garrido M, Tanimoto N, et al. Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia. Hum Mol Genet. 2016. pmid:26740549.
  24. 24. Paquet-Durand F, Sanges D, McCall J, Silva J, van Veen T, Marigo V, et al. Photoreceptor rescue and toxicity induced by different calpain inhibitors. J Neurochem. 2010;115(4):930–40. pmid:20807308.
  25. 25. Paquet-Durand F, Silva J, Talukdar T, Johnson LE, Azadi S, van Veen T, et al. Excessive activation of poly(ADP-ribose) polymerase contributes to inherited photoreceptor degeneration in the retinal degeneration 1 mouse. J Neurosci. 2007;27(38):10311–9. pmid:17881537.
  26. 26. Paquet-Durand F, Azadi S, Hauck SM, Ueffing M, van Veen T, Ekstrom P. Calpain is activated in degenerating photoreceptors in the rd1 mouse. J Neurochem. 2006;96(3):802–14. pmid:16405498.
  27. 27. Nakazawa M, Suzuki Y, Ito T, Metoki T, Kudo T, Ohguro H. Long-term effects of nilvadipine against progression of the central visual field defect in retinitis pigmentosa: an extended study. Biomed Res Int. 2013;2013:585729. pmid:24319686; PubMed Central PMCID: PMCPMC3844269.
  28. 28. Nakazawa M, Ohguro H, Takeuchi K, Miyagawa Y, Ito T, Metoki T. Effect of nilvadipine on central visual field in retinitis pigmentosa: a 30-month clinical trial. Ophthalmologica. 2011;225(2):120–6. pmid:20948238.
  29. 29. Liu X, Kerov V, Haeseleer F, Majumder A, Artemyev N, Baker SA, et al. Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2. Channels (Austin). 2013;7(6):514–23. pmid:24064553; PubMed Central PMCID: PMCPMC4042486.
  30. 30. Zabouri N, Haverkamp S. Calcium channel-dependent molecular maturation of photoreceptor synapses. PLoS One. 2013;8(5):e63853. pmid:23675510; PubMed Central PMCID: PMCPMC3652833.