MIR96 Has Good Potential to Differentiate Human Bone Marrow-Derived Mesenchymal Stem Cells into Photoreceptor-Like Cells.

OBJECTIVES: MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. MATERIALS AND METHODS: A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. CONCLUSIONS: Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina.

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin.

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.

Legumain affects the PI3K/AKT tumor progression pathway in retinoblastoma.

Known as a common malignant tumor among children, retinoblastoma (RB) is highly malignant and has poor prognosis, damages children's vision and degrades quality of life. To identify a potential molecular mechanism of RB, we conducted this study on legumain (LGMN), which is highly expressed in multiple tumors. In this study, we found that LGMN was significantly upregulated in RB cells and was positively expressed in RB tissues. We confirmed that LGMN overexpression (LGMN-OE) can promote RB cell proliferation and inhibit cell apoptosis through CCK8 experiments and flow cytometry. In addition, real-time quantitative polymerase chain reaction (RT‒qPCR) and Western blot results showed that LGMN-OE could regulate the expression of epithelial-mesenchymal transformation-related genes and proteins, related to tumor invasion and metastasis. Moreover, after LGMN knock down, the result was the opposite., RNA sequence analysis revealed 1159 differentially expressed genes between LGMN-OE and the negative control (NCOE), of which 564 were upregulated and 595 were downregulated. The first 10 genes were verified by RT‒qPCR based on P value and fold change. Interestingly, we found that LGMN could regulate the expression of recoverin (RCVRN)through a gene responsible for cancer-related retinopathy. We also screened and verified that LGMN partially activated the PI3K/AKT pathway in RB. Furthermore, we evaluated the effect of legumain inhibitors (e.g., esomeprazole) on RB, and the results suggest that esomeprazole may provide a reference for the clinical adjuvant treatment of RB. In conclusion, legumain can serve as an attractive target for RB therapy and hopefully provide new insights and ideas for the development of targeted drugs and precise personalized clinical therapy.

Iris-derived induced pluripotent stem cells that express GFP in all somatic cells of mice and differentiate into functional retinal neurons.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes.

Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma.

Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells.

Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.

IGFBPs mediate IGF-1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids.

Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors.

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.

NRL-/- gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors.

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL-/- ), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL-/- OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL-/- OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.

Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Effects of Membrane and Biological Target on the Structural and Allosteric Properties of Recoverin: A Computational Approach.

Recoverin (Rec) is a prototypical calcium sensor protein primarily expressed in the vertebrate retina. The binding of two Ca2+ ions to the functional EF-hand motifs induces the extrusion of a myristoyl group that increases the affinity of Rec for the membrane and leads to the formation of a complex with rhodopsin kinase (GRK1). Here, unbiased all-atom molecular dynamics simulations were performed to monitor the spontaneous insertion of the myristoyl group into a model multicomponent biological membrane for both isolated Rec and for its complex with a peptide from the GRK1 target. It was found that the functional membrane anchoring of the myristoyl group is triggered by persistent electrostatic protein-membrane interactions. In particular, salt bridges between Arg43, Arg46 and polar heads of phosphatidylserine lipids are necessary to enhance the myristoyl hydrophobic packing in the Rec-GRK1 assembly. The long-distance communication between Ca2+-binding EF-hands and residues at the interface with GRK1 is significantly influenced by the presence of the membrane, which leads to dramatic changes in the connectivity of amino acids mediating the highest number of persistent interactions (hubs). In conclusion, specific membrane composition and allosteric interactions are both necessary for the correct assembly and dynamics of functional Rec-GRK1 complex.

Molecular Recognition of Rhodopsin Kinase GRK1 and Recoverin Is Tuned by Switching Intra- and Intermolecular Electrostatic Interactions.

G protein-coupled receptor kinase 1 (GRK1) or rhodopsin kinase is under specific control of the neuronal Ca2+-sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation-π interaction pair in the recoverin-GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca2+-dependent membrane binding, and protein-protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent binding of recoverin to GRK1, reversing the charge from K to E led to more distortions in the interaction process, but both mutations increased the stability of the protein conformation. Molecular dynamics simulations provided an explanation for these findings as they let us suggest that residue 192 per se is not a major stabilizer of the interaction between recoverin and its target but rather that the native K is involved in a network of switching electrostatic interactions in wild-type recoverin.

Experimental Insight into the Structural and Functional Roles of the 'Black' and 'Gray' Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs.

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I ('black' cluster) and cluster II ('grey' cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two 'black' clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two 'gray' clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the 'black' clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the 'gray' cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A ('black' cluster) as well as W156A and K119A ('gray' cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.

Binding of a Myristoylated Protein to the Lipid Membrane Influenced by Interactions with the Polar Head Group Region.

Many cytoplasmic proteins contain a hydrophobic acyl chain, which facilitates protein binding to cell membranes. Hydrophobic interactions between the exposed acyl chain of the protein and hydrocarbon chains of lipids in the cell membrane are the driving force for this specific lipid-protein interaction. Recent studies point out that in addition to hydrophobic interactions the charge-charge and charge-dipole interactions between the polar head groups and basic amino acids contribute significantly to the binding process. Recoverin possesses a myristoyl chain at the N-terminus. In the presence of Ca2+ ions, the protein undergoes structural rearrangements, leading to the extrusion of the myristoyl chain, facilitating the protein binding to the membrane. In this work, we investigate the impact of interactions between the polar head group region of lipid molecules and recoverin which binds to the model membrane. The interaction with a planar lipid bilayer composed of phosphatidylcholine and cholesterol with myristoylated and nonmyristoylated recoverin is studied by in situ polarization modulation infrared reflection absorption spectroscopy. The binding of recoverin to the lipid bilayer depends on the transmembrane potential, indicating that the orientation of the permanent surface dipole in the supramolecular assembly of the lipid membrane influences the protein attachment to the membrane surface. Analysis of the amide I' mode indicates that the orientation of recoverin bound to the lipid bilayer is independent of the presence of myristoyl chain in the protein and of the folding of the protein into the tense or relaxed state. In contrast, it changes as a function of the membrane potential. At positive transmembrane potentials, the α-helical fragments of recoverin are oriented predominantly parallel to the bilayer surface. This orientation facilitates the insertion of the acyl chain of the protein into the hydrophobic region of the bilayer. At negative transmembrane potentials, the α-helical fragments of recoverin change their orientation with respect to the membrane surface, which is followed by the removal of the myristoyl chain from the membrane.

Investigating the Ca2+-dependent and Ca2+-independent mechanisms for mammalian cone light adaptation.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.

BACKGROUND: Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases. METHODS: Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing. RESULTS: Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively. CONCLUSIONS: Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.

A Highly Compensated Interferometer for Biochemical Analysis.

Here we report an improved interferometric sensing approach that facilitates high sensitivity nanovolume refractive index (RI) measurements and molecular interaction assays without a temperature controller. The compensated backscattering interferometer (CBSI) is based on a helium-neon (He-Ne) laser, a microfluidic chip, and a CCD array. The CBSI enables simultaneous differential RI measurements within nanoliter volumes, at a compensation level of ca. 5 × 10-8 RIU in the presence of large thermal perturbations (8 °C). This level of d n/d T compensation is enabled by elongating the laser beam along the central axis of the microfluidic channel and measuring the difference in positional shift of interference patterns from two adjacent regions of the channel. By separating two solutions by an air gap or oil droplet, CBSI can discriminate the difference in RI for the sample and reference at a detection limit of 7 × 10-7 RIU in the absence of electronic filtering. At this level of ΔRI sensitivity, it is possible to perform label-free, free-solution biochemical assays at the 10s of nM level without the typical high-resolution temperature control needed in conventional interferometers. Here we illustrate the effective use of CBSI by quantifying the binding affinities for mannose-concanavalin A and Ca2+-recoverin interactions.

Novel approaches to probe the binding of recoverin to membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.