Crumbs homologue-1 or CRB1 proteins are localized in Müller glia cells. They regulate cell polarization and prevent the loss of adhesion between Müller glia cells and photoreceptors. The CRB1 protein has a single short transmembrane domain, a short 37 amino acid carboxy-terminal end in the cytosol, and a long extracellular protein domain. The protein is localized at a subapical region (SAR) adjacent to adherens junctions (AJs) between photoreceptors and Müller glia cells at the outer limiting membrane (OLM) of the retina. Photoreceptors and Müller glia cells are held together by cadherins at the adherens junctions, with presumed functions for CRB1, CRB2 and CRB3 in indirectly regulating the adherens function of cadherins. We have generated mouse models for Crb1-associated retinal degeneration. Crb1 knock-out and knock-in mice are completely normal except for onset of retinal degeneration. We use these mice to test the efficacy of gene therapy vectors to prevent the onset of retinal degeneration.

The PALS1 or MPP5 protein, a membrane associated guanylate kinase (MAGUK), plays an important role in correctly localizing CRB1, CRB2, CRB3, MUPP1, and VELI3 at the subapical region (SAR) adjacent to adherens junctions (AJs) at the outer limiting membrane (OLM). Other MAGUK proteins are present in the retina as well. These are e.g. the photoreceptor specific MPP4 and the ubiquitously expressed MPP3 at the OLM. The physiological roles of MPP3, MPP4, and PALS1 have yet to be revealed. We have generated mice that lack MPP4 and showed that MPP4 is essential for correct localization of PSD95 and VELI3 at the outer plexiform layer (OPL) of the retina. We will also sort out possible overlapping and unique physiological functions, and the mechanisms where these proteins are involved in.

We use standard and Cre-loxP homologous recombination technology to generate mouse models for eye disease. These methods are reliable but however also time consuming and expensive. A recent advance has been the finding that introduction of short interfering RNAs (siRNAs) into mammalian cells can effectively silence expression of specific genes through a process known as RNA interference. In order to stably silence expression, we use adapted mammalian expression vectors originally developed at The Netherlands Cancer Institute that direct the synthesis of short hairpin RNAs (shRNAs). We have successfully used such vectors to stably silence expression of genes in primary retina cell culture. We used it e.g. to investigate the function of PALS1, a CRB1-associated protein, in retina. The shRNA vectors will allow us for the first time to systematically analyze the function of each protein in the CRB1 protein complex.

The groups of Joost Verhaagen (NIN) and Robin Ali (University College London, Institute of Ophthalmology, London, UK), have excellent expertise in gene therapy techniques. Our group collaborates with them to develop adeno-associated viral cDNA expression vectors suitable to target CRB1-deficient Müller glia cells. These vectors are being used to determine their suitability to stop or prevent retinal degeneration in mouse models for LCA and RP. Electrophysiological tests for retinal function are performed in collaboration with the group of Mathias Seeliger (Retinal Electrodiagnostics Research Group, Department of Ophthalmology, University of Tübingen).

The 6 Members of the EC Crumbs In Sight consortium are:
Elisabeth Knust, Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany. André Le Bivic, IBDM Faculte des Sciences de Luminy, CNRS, Marseille, France. Pen Rashbass, University of Sheffield, United Kingdom. Mathias Seeliger, University of Tuebingen, Germany. Sander van Deventer , Amsterdam Molecular Therapeutics. Jan Wijnholds, Netherlands Institute for Neuroscience, KNAW, Amsterdam (coordinator)

with contributions by:

Vania Broccoli, Stem Cell Research Institute San Raffaele Hospital, Milan, Italy.

Frans Cremers , Radboud University Nijmegen Medical Center, The Netherlands.

Other collaborations are ongoing with several groups in Europe and the USA.

Future Projects
We will focus on the dynamics of polarity and adhesion complex formation and maintenance. Our future projects will be dedicated to biomedical research on retinal and neuronal degeneration that significantly contribute to the development of therapeutic traits.

Goals
To understand the mechanisms that underlie deregulated cell polarization and adhesion in retinal degeneration and stimulate to apply this knowledge in the development of therapeutic tools.