Alisdair R. Philp

A knockin mouse model of the Bardet–Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity

Bardet–Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for ≈80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1M390R/M390R knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2−/−, Bbs4−/−, and Bbs6−/− mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.

Regulation of gene expression in the mammalian eye and its relevance to eye disease

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Visual Impairment in the Absence of Dystroglycan

Ocular involvement in muscular dystrophy ranges from structural defects to abnormal electroretinograms. While the mechanisms underlying the abnormal retinal physiology in patients are not understood, it is thought that α-dystroglycan extracellular interactions are critical for normal visual function. Here we show that β-dystroglycan anchors dystrophin and the inward rectifying K+ channel Kir4.1 at glial endfeet and that disruption of dystrophin and potassium channel clustering in dystroglycan mutant mice is associated with an attenuation of the electroretinogram b-wave. Glial-specific inactivation of dystroglycan or deletion of the cytoplasmic domain of β-dystroglycan was sufficient to attenuate the electroretinogram b-wave. Unexpectedly, deletion of the β-dystroglycan cytoplasmic domain did not disrupt the laminar structure of the retina. In contrast to the role of α-dystroglycan extracellular interactions during early development of the CNS, β-dystroglycan intracellular interactions are important for visual function but not the laminar development of the retina.

Novel mutations in BBS5 highlight the importance of this gene in non-Caucasian Bardet–Biedl syndrome patients

Tina Duelund Hjortshøj,* Karen Grønskov, Alisdair R. Philp, Darryl Y. Nishimura, Adebowale Adeyemo, Charles N. Rotimi, Val C. Sheffield, Thomas Rosenberg, and Karen Brøndum-Nielsen Kennedy Center, Medical Genetics Laboratory, Glostrup, Denmark Department of Paediatrics, Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa Department of Ophthalmology, Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa National Human Genome Center, Howard University, Washington, District of Columbia Kennedy Center, National Eye Clinic, Hellerup, Denmark

Visual function testing: A quantifiable visually guided behavior in mice

A measure of improved vision remains the most meaningful way to demonstrate the efficacy of a therapy. Animal models allow us to describe the pathology of inherited retinal degenerations and to evaluate emerging therapies in specific disorders in ways not possible in human subjects. The potential use of mice in this role has been limited by the lack of a simple, unambiguous and practical test of an innate visually guided behavior. To begin to address this need, we have developed equipment and protocols to measure a performance enhancing effect of vision on use of a running wheel; a scotopic visually guided behavior termed positive masking. This assay is objective, quantitative, automated and can be adapted for in-depth studies of visual thresholds, longitudinal studies of visual pathology or treatment efficacy, and large scale screening programs. Proof-of-principle experiments show that our equipment and protocols are able to characterize the full range of masking responses in normal mice in an informative and efficient manner. A sustained activity increase across a range of dim light irradiances was consistent with scotopic visual guidance of behavior, while at higher irradiances a dose dependent suppression of activity was apparent. This study also describes for the first time the interaction of experience and vision in performing a task. Specifically, we identified an experience dependent acclimatization to wheel use in scotopic conditions; a performance reduction in complete darkness; and a partial but not complete recovery of performance levels with experience in complete darkness. This suggests that where visual guidance is performance enhancing but not essential, loss of the contribution of visual guidance to the tasks might be compensated for by experience or training.

Divergent phenotypes of vision and accessory visual function in mice with visual cycle dysfunction (Rpe65 rd12) or retinal degeneration (rd/rd).

PURPOSE Tests of vision for mice remain limited and the visual phenotype of some retinal disorders in mice remain poorly understood. A novel assay of vision was used to determine how the form and extent of retinal disease affects visual phenotype in mice. METHODS Retinal histology, the suppression of locomotion by light and visual guidance of locomotion, were assessed in mice with progressive photoreceptor degeneration (rd/rd) or visual cycle dysfunction (Rpe65 rd12). RESULTS In wild-type mice, there was visual guidance of locomotor activity in dim light and suppression of activity (negative masking) in bright light. In rd/rd mice, vision was sufficient to guide locomotion at postnatal day (P)34 but was lost from P46 onward. In bright light rd/rd mice had enhanced negative masking. Although Rpe65 rd12 mice had no dim light response, with high illumination, vision was sufficient to guide locomotion at all ages tested. CONCLUSIONS A major concern for gene and cell replacement therapies is the development of visual pathways through which restored retinal function can connect to visual centers of the brain. The residual retinal response to high illumination in Rpe65 rd12 mice translates into useful vision, and visual pathways remain functional--a prerequisite for restoring vision in disorders of the retina. Similarly, useful vision in young rd/rd mice shows that there is visual pathway function before photoreceptor degeneration and suggests the potential for early therapy. Together, these findings recommend observation of masking responses in the assessment of gene and cell replacement therapies.