Nobushige Tanaka

Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.

Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin.

The rod and cone cells of the mammalian retina are the principal photoreceptors for image-forming vision. They transmit information by means of a chain of intermediate cells to the retinal ganglion cells, which in turn send signals from the retina to the brain. Loss of photoreceptor cells, as happens in a number of human diseases, leads to irreversible blindness. In a mouse model (rd/rd) of photoreceptor degeneration, we used a viral vector to express in a large number of retinal ganglion cells the light sensitive protein melanopsin, normally present in only a specialized subset of the cells. Whole-cell patch–clamp recording showed photoresponses in these cells even after degeneration of the photoreceptors and additional pharmacological or Cd2+ block of synaptic function. Interestingly, similar responses were observed across a wide variety of diverse types of ganglion cell of the retina. The newly melanopsin-expressing ganglion cells provided an enhancement of visual function in rd/rd mice: the pupillary light reflex (PLR) returned almost to normal; the mice showed behavioral avoidance of light in an open-field test, and they could discriminate a light stimulus from a dark one in a two-choice visual discrimination alley. Recovery of the PLR was stable for at least 11 months. It has recently been shown that ectopic retinal expression of a light sensitive bacterial protein, channelrhodopsin-2, can restore neuronal responsiveness and simple visual abilities in rd/rd mice. For therapy in human photodegenerations, channelrhodopsin-2 and melanopsin have different advantages and disadvantages; both proteins (or modifications of them) should be candidates.

Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy

Lentiviral vectors have become attractive delivery vehicles for gene therapy investigators. Specifically, the ability of lentiviral vectors to integrate into nondividing cells and provide stable and long-term gene expression in vivo is a desirable attribute of gene therapy approaches. We report here a simple method for generating transgenic mice using lentiviral vectors, which could be useful models for gene therapy. After removal of the zona pellucida, fertilized eggs were co-incubated with oncoretroviral or lentiviral vectors. The resulting blastocysts were transferred into uteri of pseudo-pregnant females. In both cases, around 60-70% of founder pups were transgenic as determined by PCR analysis. Southern blot analysis revealed that the transgenes were integrated at different genetic loci and transmitted through the germ line. Most of the transgenes delivered by lentiviral vectors were expressed in transgenic mice, although those delivered by oncoretroviral vectors were completely silenced. When the upstream sequences of the rhodopsin gene and the red pigment gene were used as tissue-specific promoters, consistent enhanced green fluorescent protein (EGFP) expression was observed in rod and cone photoreceptor cells, respectively, in retina. However, mice generated with the corneal epithelium-specific keratin-12 promoter displayed EGFP expression not only in cornea but also in other tissues of the mouse. We conclude that the generation of transgenic mice using lentiviral vectors is a simple and robust method to evaluate the promoter specificity in lentiviral vectors in vivo prior to undertaking a gene therapy strategy.

Reciprocity between phase shifts and amplitude changes in the mammalian circadian clock

Circadian rhythms help organisms adapt to predictable daily changes in their environment. Light resets the phase of the underlying oscillator to maintain the organism in sync with its surroundings. Light also affects the amplitude of overt rhythms. At a critical phase during the night, when phase shifts are maximal, light can reduce rhythm amplitude to nearly zero, whereas in the subjective day, when phase shifts are minimal, it can boost amplitude substantially. To explore the cellular basis for this reciprocal relationship between phase shift and amplitude change, we generated a photoentrainable, cell-based system in mammalian fibroblasts that shares several key features of suprachiasmatic nucleus light entrainment. Upon light stimulation, these cells exhibit calcium/cyclic AMP responsive element-binding (CREB) protein phosphorylation, leading to temporally gated acute induction of the Per2 gene, followed by phase-dependent changes in phase and/or amplitude of the PER2 circadian rhythm. At phases near the PER2 peak, photic stimulation causes little phase shift but enhanced rhythm amplitude. At phases near the PER2 nadir, on the other hand, the same stimuli cause large phase shifts but dampen rhythm amplitude. Real-time monitoring of PER2 oscillations in single cells reveals that changes in both synchrony and amplitude of individual oscillators underlie these phenomena.

Unfolding characteristics of a new hydrophobic acrylic intraocular lens, and possible association with complications in triple procedures

Background:  To evaluate the relationship between complications observed in triple procedures and the unfolding characteristics of the HOYA AF‐1 lens.

Bilateral subretinal haemorrhage with Terson's syndrome

PurposeTo present a case of Tersons syndrome with bilateral subretinal haemorrhage emanated from peripapilla, resulting in Mariotte blind spot enlargement.MethodsPreoperative CT scan and postoperative eye examinations, including funduscopy, fluorescein angiography, optical coherence tomography (OCT), and Goldmann perimetry.ResultsA 41-year-old Japanese man had suffered a subarachnoid haemorrhage. Three months later, he recovered from disturbance of consciousness and was referred for decreased vision in both eyes. A CT scan, obtained on the day after the event, had revealed bilateral vitreous hemorrhage. The patient underwent a standard pars plana vitrectomy to clear vitreous haemorrhage. Surprisingly we found bilateral subretinal haemorrhage around peripapilla during surgery. Although subretinal haemorrhage was almost absorbed at six months after the operation, Mariotte blind spot enlargement corresponding to the area of subretinal haemorrhage was detected in both eyes.ConclusionsIn some population of the patients with Tersons syndrome, it was demonstrated that the disturbance of peripapillary structure, presumably due to intracranial hypertension, causes subretinal haemorrhage, resulting in irreversible visual field defect.

1057. Successful Gene Therapy for Retinal Degenerations by Lentiviral Gene Transfer

We have developed a lentiviral vector system that can transduce terminally differentiated retinal cells in vivo. Using this system, we designed experiments to evaluate the therapeutic potentials of gene therapy for retinal degenerations in the cyclic nucleotide-gated channel CNGA3-deficiet mice, an animal model for cone photoreceptor cell degeneration, and retinal degeneration slow (rds/rds) mice, an animal model for retinitis pigmentosa. Wild type human CNGA3 and mouse Peripherin cDNAs were transduced in cone and rod photoreceptor cell-specific manner by human red pigment gene and bovine rhodopsin gene promoters in CNGA3-deficient mice and rds/rds mice, respectively. Moreover the additional neuroprotective effects of human pigment epithelium-derived factor (hPEDF) driven by cytomegalovirus promoter were also assessed. In CNGA3-deficient mice, transduction of hCNGA3 and hPEDF reduced the speed of cone degeneration, although subacute thinning of outer nuclear layer (ONL) was observed in some samples. Electrophysiological analysis revealed that hCNGA3 reconstitution improved cone response and maximal response which is derived from rods and cones. These effects were prolonged by combinational transduction of hPEDF. In rds/rds mice, transduction of mPeripherin and hPEDF partially prevented thinning of ONL until eight months after the injection. Electrophysiologically we could detect improvement of rod response and maximal response, although the additional effects of hPEDF were not observed. We conclude that subretinal lentiviral vector delivery had significant rescue effects on these two retinal degeneration animal models.