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Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.

Liu X, Kerov V, Haeseleer F, Majumder A, Artemyev N, Baker SA, Lee A - Channels (Austin) (2013)

Bottom Line: Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina.However, after postnatal day 13, many PR ribbons retain the immature morphology.Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics; University of Iowa; Iowa City, IA USA; Departments of Otolaryngology-Head and Neck Surgery, and Neurology; University of Iowa; Iowa City, IA USA.

ABSTRACT
Mutations in the gene encoding Cav 1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav 1.4 channels, there are defects in the development of "ribbon" synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav 1.4 channels. Whether defects in PR synapse development due to altered Cav 1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav 1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav 1.4 activation is either enhanced (Cav 1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav 1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

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Figure 4. The maturation of synaptic ribbons is impaired in CaBP4 KO and Cav1.4I756T mouse retina. (A–C) Immunofluorescence for RIBEYE (red) and Cav1.4 (green) in retina from WT (A), CaBP4 KO (B), and Cav1.4I756T (C) mice (P5-P15). Mature (arrows) and immature (arrowheads) ribbon morphologies are indicated. Scale bars: 2 μm. (D) Quantitative analysis of ribbon morphologies in retinal sections that were single-labeled with RIBEYE antibodies. RIBEYE-positive profiles were categorized according to morphology as described in the Materials and Methods and presented as the mean ± SEM. The total number of profiles examined was 1003 (WT), 1073 (Cav1.4I756T), 1140 (CaBP4 KO), and 1118 (Cav1.4 KO) in 5 animals per genotype; the retinas from each were processed in independent experiments. *, p < 0.001 compared with WT by ANOVA; #, p < 0.05 compared with CaBP4 KO by Student Newman-Keuls test.
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Figure 4: Figure 4. The maturation of synaptic ribbons is impaired in CaBP4 KO and Cav1.4I756T mouse retina. (A–C) Immunofluorescence for RIBEYE (red) and Cav1.4 (green) in retina from WT (A), CaBP4 KO (B), and Cav1.4I756T (C) mice (P5-P15). Mature (arrows) and immature (arrowheads) ribbon morphologies are indicated. Scale bars: 2 μm. (D) Quantitative analysis of ribbon morphologies in retinal sections that were single-labeled with RIBEYE antibodies. RIBEYE-positive profiles were categorized according to morphology as described in the Materials and Methods and presented as the mean ± SEM. The total number of profiles examined was 1003 (WT), 1073 (Cav1.4I756T), 1140 (CaBP4 KO), and 1118 (Cav1.4 KO) in 5 animals per genotype; the retinas from each were processed in independent experiments. *, p < 0.001 compared with WT by ANOVA; #, p < 0.05 compared with CaBP4 KO by Student Newman-Keuls test.

Mentions: CaBP4 is a Ca2+ binding protein that interacts with, and enhances voltage-dependent activation of, Cav1.4 channels.19,20 Electroretinograms (ERGs) of CaBP4 KO mice indicate a loss of both rod and cone PR synaptic transmission,19,21 consistent with reduced function of Cav1.4 channels and the CSNB2-like phenotypes associated with human mutations in the Cabp4 gene.5,22,23 PR synapses are morphologically abnormal in CaBP4 KO mice,19 which may be due to aberrant ribbon synapse assembly and/or maturation. To probe the underlying mechanism, we analyzed developmental changes in PR ribbon morphologies in WT and CaBP4 KO mice. Compared with age-matched WT control retina, ribbons were similar in CaBP4 KO retina through P13 (Fig. 4A and B). At this age, a number of elongated and horseshoe-shaped ribbons were seen in both genotypes, which indicated no deficits in the initial stages of synapse assembly. However at P15, when all ribbons take on elongated and horseshoe-shaped morphologies in WT retina, RIBEYE-positive structures that were small and round outnumbered those resembling mature ribbons in CaBP4 KO retina (Fig. 4A and B). Double-labeling with Cav1.4 antibodies revealed the association of Cav1.4 channels with both normal and abnormal ribbons, which indicated that alterations in ribbon structure did not result from failures in Cav1.4 expression or trafficking to synapses. These findings supported a requirement for CaBP4, perhaps via regulation of Cav1.4 channels, in the maturation of rod PR ribbons.


Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.

Liu X, Kerov V, Haeseleer F, Majumder A, Artemyev N, Baker SA, Lee A - Channels (Austin) (2013)

Figure 4. The maturation of synaptic ribbons is impaired in CaBP4 KO and Cav1.4I756T mouse retina. (A–C) Immunofluorescence for RIBEYE (red) and Cav1.4 (green) in retina from WT (A), CaBP4 KO (B), and Cav1.4I756T (C) mice (P5-P15). Mature (arrows) and immature (arrowheads) ribbon morphologies are indicated. Scale bars: 2 μm. (D) Quantitative analysis of ribbon morphologies in retinal sections that were single-labeled with RIBEYE antibodies. RIBEYE-positive profiles were categorized according to morphology as described in the Materials and Methods and presented as the mean ± SEM. The total number of profiles examined was 1003 (WT), 1073 (Cav1.4I756T), 1140 (CaBP4 KO), and 1118 (Cav1.4 KO) in 5 animals per genotype; the retinas from each were processed in independent experiments. *, p < 0.001 compared with WT by ANOVA; #, p < 0.05 compared with CaBP4 KO by Student Newman-Keuls test.
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Figure 4: Figure 4. The maturation of synaptic ribbons is impaired in CaBP4 KO and Cav1.4I756T mouse retina. (A–C) Immunofluorescence for RIBEYE (red) and Cav1.4 (green) in retina from WT (A), CaBP4 KO (B), and Cav1.4I756T (C) mice (P5-P15). Mature (arrows) and immature (arrowheads) ribbon morphologies are indicated. Scale bars: 2 μm. (D) Quantitative analysis of ribbon morphologies in retinal sections that were single-labeled with RIBEYE antibodies. RIBEYE-positive profiles were categorized according to morphology as described in the Materials and Methods and presented as the mean ± SEM. The total number of profiles examined was 1003 (WT), 1073 (Cav1.4I756T), 1140 (CaBP4 KO), and 1118 (Cav1.4 KO) in 5 animals per genotype; the retinas from each were processed in independent experiments. *, p < 0.001 compared with WT by ANOVA; #, p < 0.05 compared with CaBP4 KO by Student Newman-Keuls test.
Mentions: CaBP4 is a Ca2+ binding protein that interacts with, and enhances voltage-dependent activation of, Cav1.4 channels.19,20 Electroretinograms (ERGs) of CaBP4 KO mice indicate a loss of both rod and cone PR synaptic transmission,19,21 consistent with reduced function of Cav1.4 channels and the CSNB2-like phenotypes associated with human mutations in the Cabp4 gene.5,22,23 PR synapses are morphologically abnormal in CaBP4 KO mice,19 which may be due to aberrant ribbon synapse assembly and/or maturation. To probe the underlying mechanism, we analyzed developmental changes in PR ribbon morphologies in WT and CaBP4 KO mice. Compared with age-matched WT control retina, ribbons were similar in CaBP4 KO retina through P13 (Fig. 4A and B). At this age, a number of elongated and horseshoe-shaped ribbons were seen in both genotypes, which indicated no deficits in the initial stages of synapse assembly. However at P15, when all ribbons take on elongated and horseshoe-shaped morphologies in WT retina, RIBEYE-positive structures that were small and round outnumbered those resembling mature ribbons in CaBP4 KO retina (Fig. 4A and B). Double-labeling with Cav1.4 antibodies revealed the association of Cav1.4 channels with both normal and abnormal ribbons, which indicated that alterations in ribbon structure did not result from failures in Cav1.4 expression or trafficking to synapses. These findings supported a requirement for CaBP4, perhaps via regulation of Cav1.4 channels, in the maturation of rod PR ribbons.

Bottom Line: Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina.However, after postnatal day 13, many PR ribbons retain the immature morphology.Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics; University of Iowa; Iowa City, IA USA; Departments of Otolaryngology-Head and Neck Surgery, and Neurology; University of Iowa; Iowa City, IA USA.

ABSTRACT
Mutations in the gene encoding Cav 1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav 1.4 channels, there are defects in the development of "ribbon" synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav 1.4 channels. Whether defects in PR synapse development due to altered Cav 1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav 1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav 1.4 activation is either enhanced (Cav 1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav 1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

Show MeSH
Related in: MedlinePlus