Bestrophinopathy: An RPE-Photoreceptor Interface Disease

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies. © 2017 Elsevier Lt

Canine CNGA3 Gene Mutations Provide Novel Insights Into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivoand channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivochannels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients

Complex cellular phenotype of V644del mutant channel.

<p><b>(A)</b> Cellular localization of YFP-tagged wild-type canine CNGA3 and CNGA3-V644del mutant in HEK tsA201 cells. Cells transfected with the wild-type construct showed specific fluorescence pattern of expression limited to the plasma membrane and Golgi-like organelles (arrow); an evident increase in intracellular aggregates was observed in cells transfected with V644del mutant construct consistent with abnormal trafficking and potential ER retention. Scale bar: 10μm. <b>(B)</b> cGMP- and cAMP-activated currents recorded from CNGA3-WT and a responsive patch expressing V644del mutant channels. Approximately 40% of V644del mutant patches had no cGMP-activated currents, in contrast to 100% responsive patches from the CNGA3-WT-transfected cells. This partial loss of channel activity might reflect incomplete subunit assembly associated with disruption of the coiled-coil structure as depicted in the simulation studies. The responsive patches showed cyclic nucleotide-activated currents with similar characteristics to WT channels. <b>(C)</b> Histograms of subcellular localization patterns monitored in HEK tsA201 cells co-transfected with V644del and CNGA3-WT cDNA constructs. Cells were transfected with either CNGA3-WT or CNGA3-V644del or both constructs at the indicated ratios. Each cell count represents >300 cells from at least 2 transfections (mean% ± SD).</p

CNGA3-R424W mutant channel is non-functional, but CNGA3-E306R/R424E double charge-reversal mutant rescues the phenotype.

<p><b>(A)</b> Cyclic nucleotide-activated currents of the wild-type CNGA3 and a set of R424- and E306-mutant channels recorded from excised membrane patches at -60mV and +60mV in a presence of saturating concentrations of cGMP (200μM) and cAMP (5000μM). No nucleotide-activated currents were recorded for R424W-, R424E- or E306R-mutant channels. E306R-R424E double mutant channel restored cGMP activation, producing large currents similar to the wild-type CNGA3. Detailed results from multiple patches are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s007" target="_blank">S2 Table</a>. <b>(B)</b> cGMP dose-response relationship of the wild-type and CNGA3-E306R-R424E double charge-reversal mutant channels. The plot shows the ligand concentration-dependent activation of the wild-type (black) and CNGA3 double mutant (blue) homomeric channels at -60 mV. Each data set was taken from a representative single patch. The cGMP K_0.5 is 14.2 μM for the wild-type and 118.4 μM for the mutant channel; Hill coefficient [N_h] value is 2.1 and 2.0, respectively. <b>(C)</b> Cellular localization of YFP-tagged wild-type canine CNGA3 and CNGA3-R424W mutant in HEK tsA201 cells. Cells transfected with the wild-type construct showed specific fluorescent signals in the plasma membrane and Golgi-like organelles (arrow); cells expressing the mutant protein exhibited augmented intracellular signals consistent with aggregate formation in addition to membrane and Golgi-like fluorescence. Scale bar: 10μm. <b>(D)</b> Histograms of averaged cellular localization patterns for R424- and E306-mutant constructs <i>versus</i> wild-type CNGA3. A significant increase in intracellular aggregates was found in R424W-transfected cells (red bars) <i>vs</i> CNGA3-WT (black bars), and an apparent reduction in aggregate formation in the E306R-R424E double mutant channels (blue bars). An unpaired t-test was used to compare individual mutants <i>vs</i> WT (mean% ± SD for n>300 cells). * p-value of <0.05; ** p-values <0.0001.</p

Long timescale molecular dynamics simulations of the native CLZ, V630del mutant structure, and 2:1 and 1:2 WT to V630 hybrid deletion models.

<p><b>(A)</b> Molecular dynamics trajectories of the wild-type, V630del mutant and V630 hybrid CLZ deletion models at the selected time points. The wild-type CLZ structure exhibited long timescale stability. The core structure of the coiled-coil remained intact during the entire trajectory, leading to an impressive long timescale stability and average backbone root-mean-squared deviation (RMSD) of 1.54 Å. The 2:1 WT:V630del CLZ structure showed some unfolding. The core structure of the coiled-coil retained two thirds of its wild-type structure, leading to a greater overall stability compared to the all V630del structure. However, the model did exhibit some unfolding with the V630del helix (cyan) beginning to detach from the complex. The 1:2 WT:V630del CLZ complex showed more unfolding. The core structure of the coiled-coil contains charged amino acids at opposing helical positions: 630E_a 637D_a 658K_d 665E_d 669K_a causing significant disruption at these positions. The V630del CLZ structure unfolded quickly. The core structure of the coiled-coil, now composed of destabilizing small and charged amino acids, begun to unfold shortly after initialization of the simulation. After 100 ns, most of the tertiary structure was lost, leaving an amalgam of helices and an average backbone RMSD of 9.09Å. Space filling surfaces: core <i>a</i> (pale pink) and <i>d</i> (magenta) positions applies to all panels. <b>(B)</b> Root-mean-squared deviations of the backbone structure at time t = x ns compared to the starting structure. The wild-type structure exhibits impressive stability with an average RMSD of 1.54 Å; the hybrid structures have intermediate stability with average RMSDs of 2.99 Å and 4.04 Å for the 2:1 (blue) and 1:2 (green) models, respectively. The 3-fold V630 deletion model is clearly unstable (purple) having an average backbone RMSD of 9.06 Å.</p

The V644 deletion alters CLZ core structure indicating unfolding of coiled-coil complex.

<p><b>(A)</b> Sequence alignment of the CLZ (C-terminal leucine zipper) domains in CNGA-type subunits and conservation of the V644 residue. Note that canine V644 corresponds to V630 of human CNGA3. c = canine; h = human. <b>(B)</b> Helical wheel diagrams looking down the superhelical axis of the CLZ coiled-coil from the N- to C- terminus. Heptad <i>a</i> positions are marked in pink, <i>d</i> positions are denoted in magenta, and V630 is shown in red. The V630del shifts residues from <i>b</i> and <i>e</i> heptad positions to the core <i>a</i> and <i>d</i> positions and causes the predominantly hydrophobic residues of the coiled-coil core in the wild-type structure (left) to shift destabilizing charged and small residues in the mutant complex (right). Sequence diagram of the wild-type and V630del mutant heptad is shown below. <b>(C)</b> Molecular dynamics trajectories and models of the wild-type and mutant CLZ trimeric structures. Snapshots before (t = 0 ns) and after (t = 325 ns) simulation for the wild-type CNGA3-CLZ (left). The CLZ core clearly remains intact with small perturbations near the solvent exposed N- and C- termini over long timescales. Snapshots before (t = 0 ns) and after (t = 116 ns) simulation for the V630del mutant model (right). The V630 deletion dramatically alters the previously stabilizing residue-residue interactions, leading to disruption of the CLZ coiled-coil and helical structure. Small red and blue spheres correspond to oxygen and nitrogen atoms, respectively. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s004" target="_blank">S4</a></b>and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s005" target="_blank">S5</a> Figs</b> for the full-length molecular dynamics simulations.</p

R424W mutation disrupts salt bridge interaction and destabilizes the open state of pore in a homotetrameric CNGA3 model.

<p><b>(A)</b> Schematic representation of CNGA3 subunit consisting of six transmembrane (TM) spanning segments (S1-S6) and a pore domain between S5 and S6. The highlighted last residue of S6 (blue) is the site of canine CNGA3-R424W mutation; its predicted partner, glutamic acid E306, is the first residue of S4-S5 linker. <b>(B)</b> Amino acid sequence alignment of the S4-S5 linker and S6 segment of selected shaker K^<b>+</b> channel superfamily members. The TM regions of the CNG channel family were assigned using the crystal structure of the chimeric voltage-gated potassium channel Kv1.2/2.1 (PDB ID: 2R9R). Sequence alignments of S5 domain and pore region were omitted for clarity. The R424 residue is shown in blue and its interacting partner, E306 in red. The conserved salt bridges in the Kv channels show opposite charges at these positions. c = canine, b = bovine, h = human, r = rat, m = mouse. <b>(C)</b> Side view of the wild-type CNGA3 homotetramer model and the CNGA3-R424W mutant channel equilibrated in its environment. The voltage-sensing domain (S1-S4) is presented in green, the S4-S5 linker in purple and the pore-forming region (S5-S6) in grey. The residues E306 and R424 are shown as red and blue rods, respectively. The E306:R424 interaction (wild-type) or its loss (R424W mutant) is demonstrated on the higher magnification images. Carbon atoms are labeled in cyan, nitrogens in blue and oxygens in red. Other side chains were omitted for clarity. Note that R424 forms a salt bridge with the E306 molecule in three subunits out of four. <b>(D)</b> Bottom views of the wild-type CNGA3 and CNGA3-R424W mutant channels. S6 is represented as a grey solid surface highlighting the partial closure of the pore in the R424W mutant model.</p