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Jan Wijnholds, C. Henrique Alves, Ditte M. Lundvig, Naoyuki Tanimoto, Suzanne C. Beck, Gesine Huber, Till F. Andlauer, Milan Jamrich, Mathias W. Seeliger, Bokyung Park; PALS1 In Retinal Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4353.
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The Crumbs homolog 1 gene is mutated in progressive types of autosomal recessive retinitis pigmentosa, e.g. RP Type 12, and Leber congenital amaurosis. The CRB1-membrane-associated palmitoylated protein 5 (MPP5 or PALS1) protein complex is thought to organize an intracellular protein scaffold in the retina that is involved in maintenance of photoreceptor-Müller glia cell adhesion. However, the exact function of the CRB complex in cell polarity / adhesion and photoreceptor cell survival is not well known. Therefore, to understand the physiological roles of the interacting proteins, we generated conditional knockdown (cKD), or gene silencing, mice with reduced levels of PALS1 in the retina.
Previous Pals1 gene silencing studies using an adeno-associated viral vector containing Pals1 short hairpin RNA (shRNA) in cultured retinae showed the loss of localization of CRB1, CRB2, CRB3, MUPP1 and VELI3. Using Pals1 cKD mice crossed with Rx-Cre and Nestin-Cre mice, we analyzed the retinal phenotype by electroretinography (ERG), optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) of 1, 3 and 6 month(s) old mice, and by histological and immunohistochemical (IHC) staining of E12, E16, P6, P12, P18, 1 month, 3 months, 6 months and 12 months old mice.
The ERG analysis showed that a- and b-wave response amplitudes in shPals1flox/wt;Rx-Cre/+ and shPals1 flox/wt;Nestin-Cre/+ mice were lower than in littermate control mice (Wt, shPals1, Rx-Cre/+ and Nestin-Cre/+). In OCT analysis, white, small, irregularly shaped spots were distributed at the fundus of the retina. From histological analysis, the retinal phenotype showed an irregular outer limiting membrane (OLM), half rosettes of photoreceptors in the outer nuclear layer (ONL) and outer plexiform layer (OPL), photoreceptor nuclei in the subretinal space, shortened inner and outer photoreceptor segments and a thinned outer plexiform layer. Upon analyzing the expression patterns of PALS1 associated proteins, we detected loss of localization of CRB1, CRB2, PAR3, MUPP1, glutamine synthetase and an increased expression of GFAP.
PALS1 may be a key component stabilizing the CRB-membrane-associated components near to the outer limiting membrane of the retina.
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