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Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2.

Knoflach D, Kerov V, Sartori SB, Obermair GJ, Schmuckermair C, Liu X, Sothilingam V, Garcia Garrido M, Baker SA, Glösmann M, Schicker K, Seeliger M, Lee A, Koschak A - Channels (Austin) (2013)

Bottom Line: Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated.Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter.The associated visual deficiency was substantiated in behavioral paradigms.

View Article: PubMed Central - PubMed

Affiliation: Medical University Vienna; Centre for Physiology and Pharmacology; Department of Neurophysiology and Pharmacology; Vienna, Austria.

ABSTRACT
Mutations in the CACNA1F gene encoding the Cav1.4 Ca (2+) channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family. (1) Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation-as seen in the IT gain-of-function mutant (2)-may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

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Figure 5. Expression profiles of voltage-activated Ca2+ channels in mouse retina of IT mice. The mRNA expression profiles of all high voltage-activated Ca2+ channel α1, β, and α2δ subunit isoforms were determined in retinas of adult wt (black) and IT (gray) mice. Transcript levels (i.e., number of molecules per sample; see Material and Methods) of the individual experiments were normalized to the expression of the control genes Gapdh and Sdha. Log10 transformed transcript levels were analyzed by 2-way ANOVA: genotype, F(1) = 1.6, p = 0.208; gene, F(13) = 131.5, p < 0.001; genotype*gene, F(13) = 3.3, p < 0.001; p values are derived from Holm-Sidak posthoc analysis. Data are presented as mean ± SEM. Note that in comparison to wt, expression of Cav1.4 trended to be less in IT mice (p = 0.05).
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Figure 5: Figure 5. Expression profiles of voltage-activated Ca2+ channels in mouse retina of IT mice. The mRNA expression profiles of all high voltage-activated Ca2+ channel α1, β, and α2δ subunit isoforms were determined in retinas of adult wt (black) and IT (gray) mice. Transcript levels (i.e., number of molecules per sample; see Material and Methods) of the individual experiments were normalized to the expression of the control genes Gapdh and Sdha. Log10 transformed transcript levels were analyzed by 2-way ANOVA: genotype, F(1) = 1.6, p = 0.208; gene, F(13) = 131.5, p < 0.001; genotype*gene, F(13) = 3.3, p < 0.001; p values are derived from Holm-Sidak posthoc analysis. Data are presented as mean ± SEM. Note that in comparison to wt, expression of Cav1.4 trended to be less in IT mice (p = 0.05).

Mentions: To test whether the insertion of a mutation in the CACNA1F gene induced changes in the expression of other Cav channel subunits we performed qRT-PCR experiments from adult wt and IT mice. All Cav α1 subunits except Cav1.1 were reliably expressed in IT mouse retinas, although at different expression levels (Fig. 5). Cav1.4, β2, and α2δ-4 were by far the most abundantly expressed isoforms in IT mice. Even though suggested from previous independent publications34,35 this finding has not been shown before in direct comparison using the same tissue samples. The total amount of calcium channel transcripts in IT mice was not significantly different from wt but an approximately 25% reduction in the expression of Cav1.4 (p = 0.05), β2, and α2δ-4 was evident in IT mice (Fig. 5). This finding may reflect a loss of photoreceptors, which was consistent also with our staining experiments (Fig. 2). In 2 out of 4 IT mice analyzed expression levels of the auxiliary β3 subunit were 5-fold and 20-fold higher when compared with the other wt and IT mice, respectively (data not shown). This result is a plausible explanation for the highly significant increase of β3 expression in IT mice (p < 0.001). Intriguingly, although transcripts for Cav1.3 account for only 2% of total α1 subunits in wt they were significantly higher expressed in IT (p = 0.03). The isoforms Cav2.1, β4 and α2δ-2, which have previously been shown to be co-expressed in the cerebellum25 also show comparable expression levels in the retina as implicated previously.36


Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2.

Knoflach D, Kerov V, Sartori SB, Obermair GJ, Schmuckermair C, Liu X, Sothilingam V, Garcia Garrido M, Baker SA, Glösmann M, Schicker K, Seeliger M, Lee A, Koschak A - Channels (Austin) (2013)

Figure 5. Expression profiles of voltage-activated Ca2+ channels in mouse retina of IT mice. The mRNA expression profiles of all high voltage-activated Ca2+ channel α1, β, and α2δ subunit isoforms were determined in retinas of adult wt (black) and IT (gray) mice. Transcript levels (i.e., number of molecules per sample; see Material and Methods) of the individual experiments were normalized to the expression of the control genes Gapdh and Sdha. Log10 transformed transcript levels were analyzed by 2-way ANOVA: genotype, F(1) = 1.6, p = 0.208; gene, F(13) = 131.5, p < 0.001; genotype*gene, F(13) = 3.3, p < 0.001; p values are derived from Holm-Sidak posthoc analysis. Data are presented as mean ± SEM. Note that in comparison to wt, expression of Cav1.4 trended to be less in IT mice (p = 0.05).
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Related In: Results  -  Collection

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Figure 5: Figure 5. Expression profiles of voltage-activated Ca2+ channels in mouse retina of IT mice. The mRNA expression profiles of all high voltage-activated Ca2+ channel α1, β, and α2δ subunit isoforms were determined in retinas of adult wt (black) and IT (gray) mice. Transcript levels (i.e., number of molecules per sample; see Material and Methods) of the individual experiments were normalized to the expression of the control genes Gapdh and Sdha. Log10 transformed transcript levels were analyzed by 2-way ANOVA: genotype, F(1) = 1.6, p = 0.208; gene, F(13) = 131.5, p < 0.001; genotype*gene, F(13) = 3.3, p < 0.001; p values are derived from Holm-Sidak posthoc analysis. Data are presented as mean ± SEM. Note that in comparison to wt, expression of Cav1.4 trended to be less in IT mice (p = 0.05).
Mentions: To test whether the insertion of a mutation in the CACNA1F gene induced changes in the expression of other Cav channel subunits we performed qRT-PCR experiments from adult wt and IT mice. All Cav α1 subunits except Cav1.1 were reliably expressed in IT mouse retinas, although at different expression levels (Fig. 5). Cav1.4, β2, and α2δ-4 were by far the most abundantly expressed isoforms in IT mice. Even though suggested from previous independent publications34,35 this finding has not been shown before in direct comparison using the same tissue samples. The total amount of calcium channel transcripts in IT mice was not significantly different from wt but an approximately 25% reduction in the expression of Cav1.4 (p = 0.05), β2, and α2δ-4 was evident in IT mice (Fig. 5). This finding may reflect a loss of photoreceptors, which was consistent also with our staining experiments (Fig. 2). In 2 out of 4 IT mice analyzed expression levels of the auxiliary β3 subunit were 5-fold and 20-fold higher when compared with the other wt and IT mice, respectively (data not shown). This result is a plausible explanation for the highly significant increase of β3 expression in IT mice (p < 0.001). Intriguingly, although transcripts for Cav1.3 account for only 2% of total α1 subunits in wt they were significantly higher expressed in IT (p = 0.03). The isoforms Cav2.1, β4 and α2δ-2, which have previously been shown to be co-expressed in the cerebellum25 also show comparable expression levels in the retina as implicated previously.36

Bottom Line: Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated.Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter.The associated visual deficiency was substantiated in behavioral paradigms.

View Article: PubMed Central - PubMed

Affiliation: Medical University Vienna; Centre for Physiology and Pharmacology; Department of Neurophysiology and Pharmacology; Vienna, Austria.

ABSTRACT
Mutations in the CACNA1F gene encoding the Cav1.4 Ca (2+) channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family. (1) Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation-as seen in the IT gain-of-function mutant (2)-may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

Show MeSH
Related in: MedlinePlus