Anurima Majumder

Dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2

Mutations in the gene encoding Cav1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav1.4 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 Cav1.4 channels. Whether defects in PR synapse development due to altered Cav1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav1.4-selective antibodies, we found that Cav1.4 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 Cav1.4 activation is either enhanced (Cav1.4I756T) 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 Cav1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav1.4 function for efficient PR synapse maturation, and that dysregulation of Cav1.4 channels in CSNB2 may have synaptopathic consequences.

The Recent Medicinal Chemistry Development of Jak2 Tyrosine Kinase Small Molecule Inhibitors

Since the discovery of the Jak2-V617F mutation as the causative agent in a large number of myeloproliferative neoplasms (MPNs), there has been a drive to develop Jak2 specific inhibitors that can be used in therapy for MPN patients and other Jak2-related pathologies. Over the past few years, a number of research groups have sought to develop Jak2 tyrosine kinase inhibitors. These compounds are currently in pre-clinical or clinical trials. Unfortunately, there is still a need for more potent, specific, and orally bioavailable drugs to treat these diseases. Within the past twelve months, a variety of medicinal chemistry techniques have produced several lead compounds that exhibit promising Jak2 inhibitory properties. The majority of these inhibitors target the Jak2 kinase domain in general and the ATP-binding pocket in particular. In this review, we summarize these studies and discuss the structure activity relationship (SAR) properties of several compounds. As we learn more about the key structural components that provide potency and specificity in Jak2 inhibition, we will come closer to finding suitable treatment options for individuals suffering from Jak2-mediated pathologies.

Interaction of Aryl Hydrocarbon Receptor-interacting Protein-like 1 with the Farnesyl Moiety

Background: Mutations in AIPL1, a chaperone of the lipidated visual effector phosphodiesterase-6, cause severe childhood blindness. Results: AIPL1 binds the farnesyl lipid moiety. The unique insert region of AIPL1 is critical for this interaction. Conclusion: The AIPL1-farnesyl interaction suggests its role in the interaction with phosphodiesterase-6 and normal function of AIPL1. Significance: This study describes a novel mechanism of AIPL1 in retina disease. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca2+ current, suggesting interactions of transducin with the synaptic machinery.

The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo

Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease.

Structure-function correlation of G6, a novel small molecule inhibitor of Jak2: indispensability of the stilbenoid core

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.

Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation.

Despite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation. Significance Statement Cones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones.

Extended conformation of the proline-rich domain of human aryl hydrocarbon receptor-interacting protein-like 1: implications for retina disease.

Mutations in the primate‐specific proline‐rich domain (PRD) of aryl hydrocarbon receptor‐interacting protein‐like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant cone‐rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11‐316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by small angle X‐ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506‐binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecules radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase‐6A and HSP90. We also identified the γ‐subunit of phosphodiesterase‐6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants.

A truncated form of rod photoreceptor PDE6 β-subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the γ-subunit.

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.

Expression and subcellular distribution of UNC119a, a protein partner of transducin α subunit in rod photoreceptors

A recently discovered interaction of rod transducin α subunit (Gα(t1)) with UNC119a is thought to be important for transducin trafficking in photoreceptors. In this study, we analyzed the subcellular distribution of UNC119a under different conditions of illumination in vivo. Analyses by immunofluorescence and Western blotting of retina serial tangential sections demonstrated that UNC119a resides predominantly in the rod inner segment, with a small fraction of UNC119a also appearing to infiltrate the rod outer segment. Such a distribution is consistent with the proposed role of UNC119a in facilitating transducin transport from the rod inner segment to the outer segment in the dark. In addition, UNC119a was present in smaller amounts in the cell body and synaptic region of rods. The profile of UNC119a subcellular distribution remained largely unchanged under all tested conditions of illumination, and correlated with the profile of Gα(t1) following its light-dependent translocation. Quantification by Western blotting suggested that mouse retina contains ~17 pmol of UNC119a, giving a ~1 to 4 molar ratio of UNC119a to Gα(t1). Hence, light-translocated Gα(t1) can serve as a major partner of UNC119a. Supporting this role, the levels of UNC119a were downregulated by about 2-fold in mouse retina lacking Gα(t1). As a dominant partner, Gα(t1) may potentially modulate the function of other known UNC119a-interacting proteins involved in photoreceptor ciliary trafficking and synaptic regulation, in a light-dependent manner.