Kirill A. Martemyanov

In Vitro and In Vivo Characterization of Pigment Epithelial Cells Differentiated from Primate Embryonic Stem Cells

PURPOSE To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo. METHODS Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex. RESULTS The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests. CONCLUSIONS The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.

RGS expression rate-limits recovery of rod photoresponses.

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.

Mutations in GNAL cause primary torsion dystonia

Dystonia is a movement disorder characterized by repetitive twisting muscle contractions and postures. Its molecular pathophysiology is poorly understood, in part owing to limited knowledge of the genetic basis of the disorder. Only three genes for primary torsion dystonia (PTD), TOR1A (DYT1), THAP1 (DYT6) and CIZ1 (ref. 5), have been identified. Using exome sequencing in two families with PTD, we identified a new causative gene, GNAL, with a nonsense mutation encoding p.Ser293* resulting in a premature stop codon in one family and a missense mutation encoding p.Val137Met in the other. Screening of GNAL in 39 families with PTD identified 6 additional new mutations in this gene. Impaired function of several of the mutants was shown by bioluminescence resonance energy transfer (BRET) assays.

Defects in RGS9 or its anchor protein R9AP in patients with slow photoreceptor deactivation

The RGS proteins are GTPase activating proteins that accelerate the deactivation of G proteins in a variety of signalling pathways in eukaryotes. RGS9 deactivates the G proteins (transducins) in the rod and cone phototransduction cascades. It is anchored to photoreceptor membranes by the transmembrane protein R9AP (RGS9 anchor protein), which enhances RGS9 activity up to 70-fold. If RGS9 is absent or unable to interact with R9AP, there is a substantial delay in the recovery from light responses in mice. We identified five unrelated patients with recessive mutations in the genes encoding either RGS9 or R9AP who reported difficulty adapting to sudden changes in luminance levels mediated by cones. Standard visual acuity was normal to moderately subnormal, but the ability to see moving objects, especially with low-contrast, was severely reduced despite full visual fields; we have termed this condition bradyopsia. To our knowledge, these patients represent the first identified humans with a phenotype associated with reduced RGS activity in any organ.

The R7 RGS Protein Family: Multi-Subunit Regulators of Neuronal G Protein Signaling

G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits Gβ5, an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.

Human Immunodeficiency Virus Protein Tat Induces Synapse Loss via a Reversible Process That Is Distinct from Cell Death

Human immunodeficiency virus (HIV)-1 infection of the CNS produces changes in dendritic morphology that correlate with cognitive decline in patients with HIV-1 associated dementia (HAD). Here, we investigated the effects of HIV-1 transactivator of transcription (Tat), a protein released by virus-infected cells, on synapses between hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein (PSD95–GFP). Tat (24 h) decreased the number of PSD95–GFP puncta by 50 ± 7%. The decrease was concentration-dependent (EC50 = 6 ± 2 ng/ml) and preceded cell death. Tat acted via the low-density lipoprotein receptor-related protein (LRP) because the specific LRP blocker, receptor associated protein (RAP), prevented the Tat-induced decrease in the number of PSD95–GFP puncta. Ca2+ influx through the NMDA receptor was necessary for Tat-induced synapse loss. Expression of an ubiquitin ligase inhibitor protected synapses, implicating the ubiquitin–proteasome pathway. In contrast to synapse loss, Tat induced cell death (48 h) required activation of nitric oxide synthase. The ubiquitin ligase-inhibitor nutlin-3 prevented synapse loss but not cell death induced by Tat. Thus, the pathways diverged, consistent with the hypothesis that synapse loss is a mechanism to reduce excess excitatory input rather than a symptom of the neurons demise. Furthermore, application of RAP to cultures treated with Tat for 16 h reversed synapse loss. These results suggest that the impaired network function and decreased neuronal survival produced by Tat involve distinct mechanisms and that pharmacologic targets, such as LRP, might prove useful in restoring function in HAD patients.

Absence of the RGS9·Gβ5 GTPase-activating Complex in Photoreceptors of the R9AP Knockout Mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein β subunit (Gβ5). RGS9·Gβ5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gβ5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9·Gβ5 complex, and therefore all three proteins, RGS9, Gβ5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.

Retina-Specific GTPase Accelerator RGS11/Gβ5S/R9AP Is a Constitutive Heterotrimer Selectively Targeted to mGluR6 in ON-Bipolar Neurons

Members of the R7 family of the regulators of G-protein signaling (R7 RGS) proteins form multi-subunit complexes that play crucial roles in processing the light responses of retinal neurons. The disruption of these complexes has been shown to lead to the loss of temporal resolution in retinal photoreceptors and deficient synaptic transmission to downstream neurons. Despite the well established role of one member of this family, RGS9-1, in controlling vertebrate phototransduction, the roles and organizational principles of other members in the retina are poorly understood. Here we investigate the composition, localization, and function of complexes containing RGS11, the closest homolog of RGS9-1. We find that RGS11 forms a novel obligatory trimeric complex with the short splice isoform of the type 5 G-protein β subunit (Gβ5) and the RGS9 anchor protein (R9AP). The complex is expressed exclusively in the dendritic tips of ON-bipolar cells in which its localization is accomplished through a direct association with mGluR6, the glutamate receptor essential for the ON-bipolar light response. Although association with both R9AP and mGluR6 contributed to the proteolytic stabilization of the complex, postsynaptic targeting of RGS11 was not determined by its membrane anchor R9AP. Electrophysiological recordings of the light response in mouse rod ON-bipolar cells reveal that the genetic elimination of RGS11 has little effect on the deactivation of Gαo in dark-adapted cells or during adaptation to background light. These results suggest that the deactivation of mGluR6 cascade during the light response may require the contribution of multiple GTPase activating proteins.

The membrane anchor R7BP controls the proteolytic stability of the striatal specific RGS protein, RGS9-2.

A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, Gβ5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2·Gβ5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative α-helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.

RGS6/Gβ5 complex accelerates IKACh gating kinetics in atrial myocytes and modulates parasympathetic regulation of heart rate

Rationale: The parasympathetic reduction in heart rate involves the sequential activation of m2 muscarinic cholinergic receptors (m2Rs), pertussis toxin–sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel IKACh. Molecular mechanisms regulating this critical signal transduction pathway are not fully understood. Objective: To determine whether the G protein signaling regulator Rgs6/G&bgr;5 modulates m2R-IKACh signaling and cardiac physiology. Methods and Results: Cardiac expression of Rgs6, and its interaction with G&bgr;5, was demonstrated by immunoblotting and immunoprecipitation. Rgs6−/− mice were generated by gene targeting, and the cardiac effects of Rgs6 ablation were analyzed by whole-cell recordings in isolated cardiomyocytes and ECG telemetry. Loss of Rgs6 yielded profound delays in m2R-IKACh deactivation kinetics in both neonatal atrial myocytes and adult sinoatrial nodal cells. Rgs6−/− mice exhibited mild resting bradycardia and altered heart rate responses to pharmacological manipulations that were consistent with enhanced m2R-IKACh signaling. Conclusions: The cardiac Rgs6/G&bgr;5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice.