Kerstin M. Janisch

Modulation of Phosphodiesterase6 Turnoff during Background Illumination in Mouse Rod Photoreceptors

In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase γ (PDEγ) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEγ (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEγ is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin α GTP. In PDEγ T35A rods, a change in the conformation of the PDEγ subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.

Functional Rescue of Degenerating Photoreceptors in Mice Homozygous for a Hypomorphic cGMP Phosphodiesterase 6 b Allele (Pde6bH620Q)

PURPOSE Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6alpha and PDE6beta) and two regulatory (PDE6gamma) subunits. In mice homozygous for a nonsense Pde6b(rd1) allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6b(rd1) phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6b(H620Q) was characterized further. METHODS Photoreceptor degeneration in Pde6b(H620Q) homozygotes was documented by histochemistry, whereas PDE6beta expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca(2+) recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6b(H620Q) homozygotes. RESULTS Within 3 weeks of birth, the Pde6b(H620Q) homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6beta expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6b(H620Q) homozygotes exhibited depressed rod function and elevated outer segment Ca(2+). Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors. CONCLUSIONS Pde6b(H620Q) homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca(2+) to promote photoreceptor death. As degeneration in Pde6b(H620Q) mutants is slower than in Pde6b(rd1) mice and can be suppressed by Pde6b transduction, this Pde6b(H620Q) model may provide an alternate means to explore new treatments of RP.

Removal of phosphorylation sites of γ subunit of phosphodiesterase 6 alters rod light response

The phosphodiesterase 6 γ (PDE6γ) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6γ to the transducin α subunit (Tα) initiates the hydrolysis of the second messenger cGMP, and PDE6γ in association with RGS9‐1 and the other GAP complex proteins (Gβ5, R9AP) accelerates the conversion of TαGTP to TαGDP, the rate‐limiting step in the decay of the rod light response. Several studies have shown that PDE6γ can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild‐type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6γ containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6γ could modify the time course of transduction, thereby influencing the wave form of the light response.

Neuroretinal hypoxic signaling in a new preclinical murine model for proliferative diabetic retinopathy

Diabetic retinopathy (DR) affects approximately one-third of diabetic patients and, if left untreated, progresses to proliferative DR (PDR) with associated vitreous hemorrhage, retinal detachment, iris neovascularization, glaucoma and irreversible blindness. In vitreous samples of human patients with PDR, we found elevated levels of hypoxia inducible factor 1 alpha (HIF1α). HIFs are transcription factors that promote hypoxia adaptation and have important functional roles in a wide range of ischemic and inflammatory diseases. To recreate the human PDR phenotype for a preclinical animal model, we generated a mouse with neuroretinal-specific loss of the von Hippel Lindau tumor suppressor protein, a protein that targets HIF1α for ubiquitination. We found that the neuroretinal cells in these mice overexpressed HIF1α and developed severe, irreversible ischemic retinopathy that has features of human PDR. Rapid progression of retinopathy in these mutant mice should facilitate the evaluation of therapeutic agents for ischemic and inflammatory blinding disorders. In addition, this model system can be used to manipulate the modulation of the hypoxia signaling pathways, for the treatment of non-ocular ischemic and inflammatory disorders.

Light-dependent phosphorylation of the gamma subunit of cGMP-Phophodiesterase (PDE6γ) at residue threonine 22 in intact photoreceptor neurons

The gamma subunit of rod-specific cGMP phosphodiesterase 6 (PDE6gamma), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6gamma on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6gamma may increase or decrease the ability of PDE6gamma to deactivate phototransduction. To resolve role of phosphorylation of PDE6gamma in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6gamma (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6gamma, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6gamma is essential for the regulation of G-protein signaling.

Cellular and Molecular Origin of Circumpapillary Dysgenesis of the Pigment Epithelium

PURPOSE We studied clinical phenotyping and TEAD1 expression in mice and humans to gain a better understanding of the primary origin in the pathogenesis of circumpapillary dysgenesis of the pigment epithelium. DESIGN Observational case series and experimental study. PARTICIPANTS Three female patients from an affected family were included for phenotypic study. Mice and human tissues were used for biochemistry and immunohistochemistry studies. METHODS We performed genetic analyses and longitudinal clinical, imaging, and electrophysiologic studies in a 3-generation family. Western blotting and immunohistochemistry were used to detect TEAD1 expression in mice and human retinal tissues. MAIN OUTCOME MEASURES Autofluorescence and optical coherence tomography (OCT) imaging were compared and reviewed from 3 patients. TEAD1 expression was compared in different tissues from mice and human samples. RESULTS A point mutation at T1261 in TEAD1 was detected in the mother. Autofluorescence and OCT imaging studies revealed choroid is involved earlier than retinal pigment epithelium (RPE). From immunoblot analysis, we discovered that TEAD1 and its cofactors YAP65 and FOXA2 are expressed in the choroid. Immunohistochemical analysis on frozen sections of mouse retina supports immunoblot results. CONCLUSIONS The primary cellular origin of circumpapillary dysgenesis of the pigment epithelium is within the choroid instead of the pigment epithelium. The loss of the RPE and photoreceptors in later stages of the disease is a secondary consequence of choroidal degeneration. Studies of the downstream targets of TEAD1 in choroidal cells will provide promising new research opportunities for the development of treatments for choroidal diseases. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.