Aland Island Eye Disease with Retinoschisis in the Clinical Spectrum of CACNA1F-Associated Retinopathy-A Case Report.

Aland island eye disease (AIED), an incomplete form of X-linked congenital stationary night blindness (CSNB2A), and X-linked cone-rod dystrophy type 3 (CORDX3) display many overlapping clinical findings. They result from mutations in the CACNA1F gene encoding the α1F subunit of the Cav1.4 channel, which plays a key role in neurotransmission from rod and cone photoreceptors to bipolar cells. Case report: A 57-year-old Caucasian man who had suffered since his early childhood from nystagmus, nyctalopia, low visual acuity and high myopia in both eyes (OU) presented to expand the diagnostic process, because similar symptoms had occurred in his 2-month-old grandson. Additionally, the patient was diagnosed with protanomalous color vision deficiency, diffuse thinning, and moderate hypopigmentation of the retina. Optical coherence tomography of the macula revealed retinoschisis in the right eye and foveal hypoplasia in the left eye. Dark-adapted (DA) 3.0 flash full-field electroretinography (ffERG) amplitudes of a-waves were attenuated, and the amplitudes of b-waves were abolished, which resulted in a negative pattern of the ERG. Moreover, the light-adapted 3.0 and 3.0 flicker ffERG as well as the DA 0.01 ffERG were consistent with severely reduced responses OU. Genetic testing revealed a hemizygous form of a stop-gained mutation (c.4051C>T) in exon 35 of the CACNA1F gene. This pathogenic variant has so far been described in combination with a phenotype corresponding to CSNB2A and CORDX3. This report contributes to expanding the knowledge of the clinical spectrum of CACNA1F-related disease. Wide variability and the overlapping clinical manifestations observed within AIED and its allelic disorders may not be explained solely by the consequences of different mutations on proteins. The lack of distinct genotype-phenotype correlations indicates the presence of additional, not yet identified, disease-modifying factors.

A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.

Mouse all-cone retina models of Cav1.4 synaptopathy.

The voltage-gated calcium channel, Cav1.4 is localized to photoreceptor ribbon synapses and functions both in molecular organization of the synapse and in regulating release of synaptic vesicles. Mutations in Cav1.4 subunits typically present as either incomplete congenital stationary night blindness or a progressive cone-rod dystrophy in humans. We developed a cone-rich mammalian model system to further study how different Cav1.4 mutations affect cones. RPE65 R91W KI; Nrl KO "Conefull" mice were crossed to Cav1.4 α1F or α2δ4 KO mice to generate the "Conefull:α1F KO" and "Conefull:α2δ4 KO" lines. Animals were assessed using a visually guided water maze, electroretinogram (ERG), optical coherence tomography (OCT), and histology. Mice of both sexes and up to six-months of age were used. Conefull: α1F KO mice could not navigate the visually guided water maze, had no b-wave in the ERG, and the developing all-cone outer nuclear layer reorganized into rosettes at the time of eye opening with degeneration progressing to 30% loss by 2-months of age. In comparison, the Conefull: α2δ4 KO mice successfully navigated the visually guided water maze, had a reduced amplitude b-wave ERG, and the development of the all-cone outer nuclear layer appeared normal although progressive degeneration with 10% loss by 2-months of age was observed. In summary, new disease models for studying congenital synaptic diseases due to loss of Cav1.4 function have been created.

Cav1.4 congenital stationary night blindness is associated with an increased rate of proteasomal degradation.

Pathogenic, generally loss-of-function, variants in CACNA1F, encoding the Cav1.4α1 calcium channel, underlie congenital stationary night blindness type 2 (CSNB2), a rare inherited retinal disorder associated with visual disability. To establish the underlying pathomechanism, we investigated 10 clinically derived CACNA1F missense variants located across pore-forming domains, connecting loops, and the carboxy-tail domain of the Cav1.4α subunit. Homology modeling showed that all variants cause steric clashes; informatics analysis correctly predicted pathogenicity for 7/10 variants. In vitro analyses demonstrated that all variants cause a decrease in current, global expression, and protein stability and act through a loss-of-function mechanism and suggested that the mutant Cav1.4α proteins were degraded by the proteasome. We showed that the reduced current for these variants could be significantly increased through treatment with clinical proteasome inhibitors. In addition to facilitating clinical interpretation, these studies suggest that proteasomal inhibition represents an avenue of potential therapeutic intervention for CSNB2.

Characterization of two pathological gating-charge substitutions in Cav1.4 L-type calcium channels.

Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.

Calmodulin promotes a Ca2+ -dependent conformational change in the C-terminal regulatory domain of CaV 1.2.

Calmodulin (CaM) binds to the membrane-proximal cytosolic C-terminal domain of CaV 1.2 (residues 1520-1669, CT(1520-1669)) and causes Ca2+ -induced conformational changes that promote Ca2+ -dependent channel inactivation (CDI). We report biophysical studies that probe the structural interaction between CT(1520-1669) and CaM. The recombinantly expressed CT(1520-1669) is insoluble, but can be solubilized in the presence of Ca2+ -saturated CaM (Ca4 /CaM), but not in the presence of Ca2+ -free CaM (apoCaM). We show that half-calcified CaM (Ca2 /CaM12 ) forms a complex with CT(1520-1669) that is less soluble than CT(1520-1669) bound to Ca4 /CaM. The NMR spectrum of CT(1520-1669) reveals spectral differences caused by the binding of Ca2 /CaM12 versus Ca4 /CaM, suggesting that the binding of Ca2+ to the CaM N-lobe may induce a conformational change in CT(1520-1669).

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

A Review of Genetic and Physiological Disease Mechanisms Associated With Cav1 Channels: Implications for Incomplete Congenital Stationary Night Blindness Treatment.

Calcium channels are crucial to a number of cellular functions. The high voltage-gated calcium channel family comprise four heteromeric channels (Cav1.1-1.4) that function in a similar manner, but that have distinct expression profiles. Three of the pore-forming α1 subunits are located on autosomes and the forth on the X chromosome, which has consequences for the type of pathogenic mutation and the disease mechanism associated with each gene. Mutations in this family of channels are associated with malignant hyperthermia (Cav1.1), various QT syndromes (Cav1.2), deafness (Cav1.3), and incomplete congenital stationary night blindness (iCSNB; Cav1.4). In this study we performed a bioinformatic analysis on reported mutations in all four Cav α1 subunits and correlated these with variant frequency in the general population, phenotype and the effect on channel conductance to produce a comprehensive composite Cav1 mutation analysis. We describe regions of mutation clustering, identify conserved residues that are mutated in multiple family members and regions likely to cause a loss- or gain-of-function in Cav1.4. Our research highlights that therapeutic treatments for each of the Cav1 channels will have to consider channel-specific mechanisms, especially for the treatment of X-linked iCSNB.

Transgenic Expression of Cacna1f Rescues Vision and Retinal Morphology in a Mouse Model of Congenital Stationary Night Blindness 2A (CSNB2A).

Purpose: Congenital stationary night blindness 2A (CSNB2A) is a genetic retinal disorder characterized by poor visual acuity, nystagmus, strabismus, and other signs of retinal dysfunction resulting from mutations in Cacna1f-the gene coding for the pore-forming subunit of the calcium channel CaV1.4. Mouse models of CSNB2A have shown that mutations causing the disease deleteriously affect photoreceptors and their synapses with second-order neurons. This study was undertaken to evaluate whether transgenic expression of Cacna1f could rescue morphology and visual function in a Cacna1f-KO model of CSNB2A. Methods: Strategic creation, breeding and use of transgenic mouse lines allowed for Cre-driven retina-specific expression of Cacna1f in a CSNB2A model. Transgene expression and retinal morphology were investigated with immunohistochemistry in retinal wholemounts or cross-sections. Visual function was assessed by optokinetic response (OKR) analysis and electroretinography (ERG). Results: Mosaic, prenatal expression of Cacna1f in the otherwise Cacna1f-KO retina was sufficient to rescue some visual function. Immunohistochemical analyses demonstrated wild-type-like photoreceptor and synaptic morphology in sections with transgenic expression of Cacna1f. Conclusions: This report describes a novel system for Cre-inducible expression of Cacna1f in a Cacna1f-KO mouse model of CSNB2A and provides preclinical evidence for the potential use of gene therapy in the treatment of CSNB2A. Translational Relevance: These data have relevance in the treatment of CSNB2A and in understanding how photoreceptor integration might be achieved in retinas in which photoreceptors have been lost, such as retinitis pigmentosa, age-related macular degeneration, and other degenerative conditions.

Mutation of an L-Type Calcium Channel Gene Leads to T Lymphocyte Dysfunction.

Calcium (Ca2+) is a vital secondary messenger in T lymphocytes regulating a vast array of important events including maturation, homeostasis, activation, and apoptosis and can enter the cell through CRAC, TRP, and CaV channels. Here we describe a mutation in the L-type Ca2+ channel CaV1.4 leading to T lymphocyte dysfunction, including several hallmarks of immunological exhaustion. CaV1.4-deficient mice exhibited an expansion of central and effector memory T lymphocytes, and an upregulation of inhibitory receptors on several T cell subsets. Moreover, the sustained elevated levels of activation markers on B lymphocytes suggest that they are in a chronic state of activation. Functionally, T lymphocytes exhibited a reduced store-operated Ca2+ flux compared to wild-type controls. Finally, modifying environmental conditions by herpes virus infection exacerbated the dysfunctional immune phenotype of the CaV1.4-deficient mice. This is the first example where the mutation of a CaV channel leads to T lymphocyte dysfunction, including the upregulation of several inhibitory receptors, hallmarks of T cell exhaustion, and establishes the physiological importance of CaV channel signaling in maintaining a nimble immune system.

Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A).

Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α1F subunit of a L-type voltage-gated calcium channel, Cav1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our 'G305X' mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function - measured by immunohistochemistry, optokinetic response and electroretinography - deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas - ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Cav1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Cav1.4 in photopic vision, it also suggests a potential new target for restorative therapy.

RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors.

Night blindness can result from impaired photoreceptor function and a subset of cases have been linked to dysfunction of Cav1.4 calcium channels and in turn compromised synaptic transmission. Here, we show that active zone proteins RIM1/2 are important regulators of Cav1.4 channel function in mouse rod photoreceptors and thus synaptic activity. The conditional double knock-out (cdko) of RIM1 and RIM2 from rods starting a few weeks after birth did not change Cav1.4 protein expression at rod ribbon synapses nor was the morphology of the ribbon altered. Heterologous overexpression of RIM2 with Cav1.4 had no significant influence on current density when examined with BaCl2 as the charge carrier. Nonetheless, whole-cell voltage-clamp recordings from cdko rods revealed a profound reduction in Ca(2+) currents. Concomitantly, we observed a 4-fold reduction in spontaneous miniature release events from the cdko rod terminals and an almost complete absence of evoked responses when monitoring changes in membrane incorporation after strong step depolarizations. Under control conditions, 49 and 83 vesicles were released with 0.2 and 1 s depolarizations, respectively, which is close to the maximal number of vesicles estimated to be docked at the base of the ribbon active zone, but without RIM1/2, only a few vesicles were stimulated for release after a 1 s stimulation. In conclusion, our study shows that RIM1/2 potently enhance the influx of Ca(2+) into rod terminals through Cav1.4 channels, which is vitally important for the release of vesicles from the rod ribbon. Significance statement: Active zone scaffolding proteins are thought to bring multiple components involved in Ca(2+)-dependent exocytosis into functional interactions. We show that removal of scaffolding proteins RIM1/2 from rod photoreceptor ribbon synapses causes a dramatic loss of Ca(2+) influx through Cav1.4 channels and a correlated reduction in evoked release, yet the channels remain localized to synaptic ribbons in a normal fashion. Our findings strongly argue that RIM1/2 facilitate Ca(2+) entry and in turn Ca(2+) evoked release by modulating Cav1.4 channel openings; however, RIM1/2 are not needed for the retention of Cav1.4 at the synapse. In summary, a key function of RIM1/2 at rod ribbons is to enhance Cav1.4 channel activity, possibly through direct or indirect modulation of the channel.

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2.

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.

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

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.

EF hand-mediated Ca- and cGMP-signaling in photoreceptor synaptic terminals.

Photoreceptors, the light-sensitive receptor neurons of the retina, receive and transmit a plethora of visual informations from the surrounding world. Photoreceptors capture light and convert this energy into electrical signals that are conveyed to the inner retina. For synaptic communication with the inner retina, photoreceptors make large active zones that are marked by synaptic ribbons. These unique synapses support continuous vesicle exocytosis that is modulated by light-induced, graded changes of membrane potential. Synaptic transmission can be adjusted in an activity-dependent manner, and at the synaptic ribbons, Ca(2+)- and cGMP-dependent processes appear to play a central role. EF-hand-containing proteins mediate many of these Ca(2+)- and cGMP-dependent functions. Since continuous signaling of photoreceptors appears to be prone to malfunction, disturbances of Ca(2+)- and cGMP-mediated signaling in photoreceptors can lead to visual defects, retinal degeneration (rd), and even blindness. This review summarizes aspects of signal transmission at the photoreceptor presynaptic terminals that involve EF-hand-containing Ca(2+)-binding proteins.