Neurodegeneration in the olfactory bulb and olfactory deficits in the Ccdc66 -/- mouse model for retinal degeneration.

The Ccdc66-deficient (Ccdc66 -/-) mouse model exhibits slow progressive retinal degeneration. It is unclear whether CCDC66 protein also plays a role in the wildtype (WT; Ccdc66 +/+) mouse brain and whether the lack of Ccdc66 gene expression in the Ccdc66 -/- mouse brain may result in morphological and behavioral alterations. CCDC66 protein expression in different brain regions of the adult WT mouse and in whole brain during postnatal development was quantified by SDS-PAGE and Western blot. Ccdc66 reporter gene expression was visualized by X-gal staining. Selected brain regions were further analyzed by light and electron microscopy. In order to correlate anatomical with behavioral data, an olfactory habituation/dishabituation test was performed. CCDC66 protein was expressed throughout the early postnatal development in the WT mouse brain. In adult mice, the main olfactory bulb exhibited high CCDC66 protein levels comparable to the expression in the retina. Additionally, the Ccdc66 -/- mouse brain showed robust Ccdc66 reporter gene expression especially in adult olfactory bulb glomeruli, the olfactory nerve layer and the olfactory epithelium. Degeneration was detected in the Ccdc66 -/- olfactory bulb glomeruli at advanced age. This degeneration was also reflected in behavioral alterations; compared to the WT, Ccdc66 -/- mice spent significantly less time sniffing at the initial presentation of unknown, neutral odors and barely responded to social odors. Ccdc66 -/- mice develop substantial olfactory nerve fiber degeneration and alteration of olfaction-related behavior at advanced age. Thus, the Ccdc66 -/- mouse model for retinal degeneration adds the possibility to study mechanisms of central nervous system degeneration.

Genome-wide profiling of S/MAR-based replicon contact sites.

Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.

Exome Sequencing Reveals AGBL5 as Novel Candidate Gene and Additional Variants for Retinitis Pigmentosa in Five Turkish Families.

PURPOSE: Retinitis pigmentosa (RP) is the most common inherited retinal disease with high genetic heterogeneity and variable phenotypes. Characteristic symptoms include night blindness and progressive loss of visual field, leading to blindness. Mutations in >60 genes have been identified to date as causative for RP, and additional candidate genes are assumed. METHODS: To find the disease-causing mutations in the affected members of five Turkish families, we sequenced whole exomes using an Illumina platform. RESULTS: Among all candidate genes for retinal degeneration we found two previously known sequence variations: a 4 bp deletion in the RPGR gene (c.1662_1665delAGAA; p.Glu555Glyfs*14) and a recently described USH1-causing missense mutation in MYO7A (c.472G>A, p.Gly158Arg). Furthermore, a novel 1 bp deletion in the VCAN gene (c.5118delA; p.Ser1707Valfs*44) was detected as well as a large deletion in EYS, spanning ∼ 400kb and comprising exons 16-26 (p.fs*). In one family, exome analyses of two affected individuals revealed a homozygous missense mutation (c.883G>A; p.Asp295Asn) in the AGBL5 (Agbl5; CCP5) gene, previously not reported to be associated with RP. RNA and protein analyses showed expression in human retina, as well as in mouse retina, brain and testis. Furthermore, cDNA analyses indicate the existence of tissue-specific AGBL5 splice variations in humans. AGBL5/CCP5 immunoreactivity was also visualized in human and mouse retinae. CONCLUSION: Due to the characteristic RP phenotype in patients carrying the AGBL5 missense mutation we suggest this gene as a candidate for a new form of autosomal recessively inherited RP and recommend further investigation to confirm this hypothesis.

Ccdc66 null mutation causes retinal degeneration and dysfunction.

Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.