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Boulling A.,University of Lausanne | Boulling A.,Institute for Research in Ophthalmology | Escher P.,University of Lausanne
Experimental Eye Research | Year: 2016

Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3rd7/rd7 retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3rd7/rd7 retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina. © 2016 Elsevier Ltd. Source

Abplanalp J.,University of Zurich | Abplanalp J.,ETH Zurich | Laczko E.,University of Zurich | Philp N.J.,Thomas Jefferson University | And 12 more authors.
Human Molecular Genetics | Year: 2013

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associatedSLC6A8. Here,weidentifieda secondcreatine transporter (CRT2)knownasmonocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration inMCT12 (p.G407S) found in a patientwith age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 μM. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels.We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventivepower of creatine supplementation against the most prominent age-related vision impaired condition. © The Author 2013. Published by Oxford University Press. All rights reserved. Source

Boisset G.,Institute for Research in Ophthalmology | Schorderet D.F.,Institute for Research in Ophthalmology | Schorderet D.F.,University of Lausanne | Schorderet D.F.,Ecole Polytechnique Federale de Lausanne
Experimental Eye Research | Year: 2012

Ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signaling molecules. Together, these molecules regulate cell proliferation, apoptosis and specify retinal fate. In the zebrafish (. Danio rerio), hmx1 is a homeobox transcription factor implicated in eye and brain development. Hmx1 transcripts were detected in the nasal retina and lens as well as otic vesicles and pharyngeal arches by 24-32 hpf. Before this stage, transcripts were more uniformly expressed in the optic vesicle. Knockdown of hmx1 led to microphthalmia. Delayed withdrawal of retinal progenitors from the cell cycle resulting in retarded retinal differentiation was observed in morphant. The retina and brain also showed an increased cell death at 24 hpf. The polarized expression of hmx1 to the nasal part in the zebrafish retina strongly suggested an involvement in the nasal-temporal patterning. However, the key patterning genes tested so far were not regulated by hmx1. Altogether, these results suggest an important role for hmx1 in retinogenesis. © 2012 Elsevier Ltd. Source

Produit-Zengaffinen N.,Institute for Research in Ophthalmology | Pournaras C.J.,University of Geneva | Schorderet D.F.,Institute for Research in Ophthalmology | Schorderet D.F.,University of Lausanne | Schorderet D.F.,Ecole Polytechnique Federale de Lausanne
Advances in Experimental Medicine and Biology | Year: 2014

The role played by autophagy after ischemia/reperfusion (I/R) in the retina remains unknown. Our study investigated whether ischemic injury in the retina, which causes an energy crisis, would induce autophagy. Retinal ischemia was induced by elevation of the intraocular pressure and modulation of autophagic markers was analyzed at the protein levels in an early and late phase of recovery. Following retinal ischemia an increase in LC3BII was first observed in the early phase of recovery but did not stay until the late phase of recovery. Post-ischemic induction of autophagy by intravitreal rapamycin administration did not provide protection against the lesion induced by the ischemic stress. On the contrary, an increase in the number of apoptotic cells was observed following I/R in the rapamycin treated retinas. © 2014, Springer Science+Business Media, LLC. Source

Allaman-Pillet N.,Institute for Research in Ophthalmology | Oberson A.,Institute for Research in Ophthalmology | Schorderet D.F.,Institute for Research in Ophthalmology | Schorderet D.F.,University of Lausanne | Schorderet D.F.,Ecole Polytechnique Federale de Lausanne
Molecular Cancer Research | Year: 2015

Retinoblastoma is the most common pediatric intraocular neoplasm. While retinoblastoma development requires the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, additional genomic changes are involved in tumor progression, which progressively lead to resistance of tumor cells to death. Therapeutics acting at very downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. The BH3-only proteins promote apoptosis by modulating the interaction between the pro- and antiapoptotic members of the BCL2 protein family, and this effect can be recapitulated by the BH3 domains. This report analyzes the effect of various BH3 peptides, corresponding to different BH3-only proteins, on two retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the photoreceptor cell line 661W. The BH3 peptide BIRO1, derived from the BCL2L11 death domain, was very effective in promoting Y79 and WERI-Rb cell death without affecting the 661W photoreceptor cells. This cell death was efficient even in absence of BAX and was shown to be caspase independent. While ROS production or AIF release was not detected from mitochondria of treated cells, BIRO1 initiated mitochondria fragmentation in a short period of time following treatment. Implications: The BIRO1 peptide is highly effective at killing retinoblastoma cells and has potential as a peptidomimetic. ©2014 AACR. Source

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