Felix Tonagel

In vivo confocal imaging of the retina in animal models using scanning laser ophthalmoscopy

Scanning-laser ophthalmoscopy is a technique for confocal imaging of the eye in vivo. The use of lasers of different wavelengths allows to obtain information about specific tissues and layers due to their reflection and transmission characteristics. In addition, fluorescent dyes excitable in the blue and infrared range offer a unique access to the vascular structures associated with each layer. In animal models, a further enhancement in specificity can be obtained by GFP expression under control of tissue-specific promotors. Important fields of application are studies in retinal degenerations and the follow-up of therapeutic intervention.

LOSS OF THE CHOLESTEROL-BINDING PROTEIN PROMININ-1/CD133 CAUSES DISK DYSMORPHOGENESIS AND PHOTORECEPTOR DEGENERATION

Prominin-1/CD133 (Prom-1) is a commonly used marker of neuronal, vascular, hematopoietic and other stem cells, yet little is known about its biological role and importance in vivo. Here, we show that loss of Prom-1 results in progressive degeneration of mature photoreceptors with complete loss of vision. Despite the expression of Prom-1 on endothelial progenitors, photoreceptor degeneration was not attributable to retinal vessel defects, but caused by intrinsic photoreceptor defects in disk formation, outer segment morphogenesis, and associated with visual pigment sorting and phototransduction abnormalities. These findings shed novel insight on how Prom-1 regulates neural retinal development and phototransduction in vertebrates.

Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia

Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.

A Single Amino Acid Substitution (Cys249Trp) in Crb1 Causes Retinal Degeneration and Deregulates Expression of Pituitary Tumor Transforming Gene Pttg1

Different mutations in the human Crumbs homolog-1 (CRB1) gene cause a variety of retinal dystrophies, such as Leber congenital amaurosis, early onset retinitis pigmentosa (e.g., RP12), RP with Coats-like exudative vasculopathy, and pigmented paravenous retinochoroidal atrophy. Loss of Crb1 leads to displaced photoreceptors and focal degeneration of all neural layers attributable to loss of adhesion between photoreceptors and Müller glia cells. To gain insight into genotype–phenotype relationship, we generated Crb1C249W mice that harbor an amino acid substitution (Cys249Trp) in the extracellular sixth calcium-binding epidermal growth factor domain of Crb1. Our analysis showed that Crb1C249W as wild-type protein trafficked to the subapical region adjacent to adherens junctions at the outer limiting membrane (OLM). Hence, these data suggest correct trafficking of the corresponding mutant CRB1 in RP12 patients. Crb1C249W mice showed loss of photoreceptors in the retina, relatively late compared with mice lacking Crb1. Scanning laser ophthalmoscopy revealed autofluorescent dots that presumably represent layer abnormalities after OLM disturbance. Gene expression analyses revealed lower levels of pituitary tumor transforming gene 1 (Pttg1) transcripts in Crb1C249W/− knock-in and Crb1−/− knock-out compared with control retinas. Exposure to white light decreased levels of Pttg1 in Crb1 mutant retinas. We hypothesize deregulation of Pttg1 expression attributable to a C249W substitution in the extracellular domain of Crb1.

Toxic optic neuropathies: an updated review

Toxic optic neuropathy (TON) is caused by the damage to the optic nerve through different toxins, including drugs, metals, organic solvents, methanol and carbon dioxide. A similar clinical picture may also be caused by nutritional deficits, including B vitamins, folic acid and proteins with sulphur‐containing amino acids. This review summarizes the present knowledge on disease‐causing factors, clinical presentation, diagnostics and treatment in TON. It discusses in detail known and hypothesized relations between drugs, including tuberculostatic drugs, antimicrobial agents, antiepileptic drugs, antiarrhythmic drugs, disulfiram, halogenated hydroquinolones, antimetabolites, tamoxifen and phosphodiesterase type 5 inhibitors and optic neuropathy.

Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice

Although Harderian glands are especially large in rodents, many features of this retroocular gland, including its development and function, are not well established. Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes expressed in this gland. PP2A substrate specificity is determined by regulatory subunits with leucine 309 of the catalytic subunit playing a crucial role in the recruitment of regulatory subunits into the complex in vitro. Here we expressed an L309A mutant catalytic subunit in Harderian gland of transgenic mice. We found a delayed postnatal development and hypoplasia of the gland, causing enophthalmos. To determine why expression of the L309A mutant caused this phenotype, we determined the PP2A subunit composition. We found an altered subunit composition in the transgenic gland that was accompanied by pronounced changes of proteins regulating cell adhesion. Specifically, cadherin and beta-catenin were dramatically reduced and shifted to the cytosol. Furthermore, we found an inactivating phosphorylation of the cadherin-directed glycogen synthase kinase-3beta. In conclusion, the carboxy-terminal leucine L309 of the PP2A catalytic subunit determines PP2A heterotrimer composition in vivo. Moreover, our data demonstrate that PP2A subunit composition plays a crucial role in regulating cell adhesion and as a consequence in the development of the Harderian gland.

Treatment of optic neuritis with erythropoietin (TONE): a randomised, double-blind, placebo-controlled trial—study protocol

Introduction Optic neuritis leads to degeneration of retinal ganglion cells whose axons form the optic nerve. The standard treatment is a methylprednisolone pulse therapy. This treatment slightly shortens the time of recovery but does not prevent neurodegeneration and persistent visual impairment. In a phase II trial performed in preparation of this study, we have shown that erythropoietin protects global retinal nerve fibre layer thickness (RNFLT-G) in acute optic neuritis; however, the preparatory trial was not powered to show effects on visual function. Methods and analysis Treatment of Optic Neuritis with Erythropoietin (TONE) is a national, randomised, double-blind, placebo-controlled, multicentre trial with two parallel arms. The primary objective is to determine the efficacy of erythropoietin compared to placebo given add-on to methylprednisolone as assessed by measurements of RNFLT-G and low-contrast visual acuity in the affected eye 6 months after randomisation. Inclusion criteria are a first episode of optic neuritis with decreased visual acuity to ≤0.5 (decimal system) and an onset of symptoms within 10 days prior to inclusion. The most important exclusion criteria are history of optic neuritis or multiple sclerosis or any ocular disease (affected or non-affected eye), significant hyperopia, myopia or astigmatism, elevated blood pressure, thrombotic events or malignancy. After randomisation, patients either receive 33 000 international units human recombinant erythropoietin intravenously for 3 consecutive days or placebo (0.9% saline) administered intravenously. With an estimated power of 80%, the calculated sample size is 100 patients. The trial started in September 2014 with a planned recruitment period of 30 months. Ethics and dissemination TONE has been approved by the Central Ethics Commission in Freiburg (194/14) and the German Federal Institute for Drugs and Medical Devices (61-3910-4039831). It complies with the Declaration of Helsinki, local laws and ICH-GCP. Trial registration number NCT01962571.

Disease mechanisms and gene therapy in a mouse model for X-linked retinoschisis.

X-linked retinoschisis (RS) is an inherited recessive macular degeneration that affects between 1 in 5000 and 1 in 25,000 males early in life (George et al., 1995; Sieving, 1998; Tantri et al., 2004). It is characterized by a loss in central vision, splitting of the retina with the appearance of spoke-like cystic cavities radiating from the parafoveal region of the retina, a loss in the b-wave of the electroretinogram (ERG), and progressive atrophy of the macula. In about 50% of the cases, bilateral schisis is observed in the peripheral retina with some loss in peripheral vision. During the course of the disease, complications can arise which include retinal detachment, vitreal hemorrhage and choroidal sclerosis.