Genetic and molecular studies of the blind-sterile (bs) and blind-sterile 2 (bs2) mouse models of cataracts and male infertility

The goal of the work presented in this dissertation was to identify novel genes involved in the formation of cataracts in mouse models, and investigate the molecular pathways involved in lens development and the maintenance of lens transparency.;Blind-sterile 2 arose at The Jackson Laboratory and was identified in a clinical screen for ophthalmic abnormalities. Initial studies mapped the mutation to mouse chromosome 2, in a region where no previously known cataract loci were located. Our additional mapping studies identified a 3.1 Mb critical region 45 cM from the centromere, containing 19 candidate genes. Sequencing of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. Agps is a peroxisomal enzyme that catalyzes an early step in the ether lipid synthesis pathway. The mutation results in two aberrantly spliced Agps transcripts which lack the catalytic FAD oxidase domain, as well as low residual levels of full-length Agps transcript. As a result, ether lipid levels in bs2 mice are significantly lower than in WT siblings. This depletion of ether lipids results in severely disrupted lens fiber cell differentiation and severe cataracts in the lens by postnatal day 28 (P28). In the testis, bs2 mice exhibit a complete absence of mature spermatozoa; however, spermatogonia and Sertoli cells are present at normal levels. Most tubules contain large multinucleate clusters of what appear to be spermatocytes which become TUNELpositive and are subsequently degraded prior to progressing to spermatid formation. In addition to the lens and testis phenotypes, bs2 mice are significantly smaller than their WT littermates at weaning age, have difficulty moving about their surroundings, and exhibit rhizomelic shortening of the humerus, which is similar to human patients with Agps mutations resulting in rhizomelic chondrodysplasia punctata (RCDP3), a disease for which bs2 was the first animal model. Current studies have identified a potential role for ether lipids in maintenance of cellular junctions such as Connexin 43 between differentiating cells in the lens which may be responsible for bs2 cataract formation.;Blind-sterile (bs) also arose spontaneously and has been utilized as a model of male infertility in a number of studies which have identified an arrest of spermatogenesis at the point of acrosome formation, and mapped the causative mutation to the distal end of mouse chromosome 2. The number of spermatogonia and Sertoli cells are unchanged in bs mice, although no cells appear to progress past the round spermatid stage and the point of acrosome formation. bs mice also exhibit an early-onset cataract phenotype by E17.5, frequently followed by the rupture of the lens capsule as early as P5. Our mapping studies further narrowed the critical region to 416 kb comprised of 16 candidate genes. Sequencing of the coding regions of all 16 genes revealed one mutation, a 12 bp deletion in the gene TBC1 domain family, member 20 (Tbc1d20). TBC1D20 is a RAB GTPase-activating protein which serves as a molecular switch regulating the signaling activity of members of the RAB family of small GTPases. The identified mutation is in a highly-conserved region of TBC1D20, and results in the protein being unstable. Our work supports a role for TBC1D20 as a RABGAP for RAB1 which regulates vesicular trafficking from the ER to the Golgi, thus serving as a negative regulator of RAB1 signaling. In bs mEFs, increased size and intensity of staining of the Golgi marker GM130 was indicative of over-active RAB1 signaling as compared to WT mEFs. In contrast, HeLa cells in which WT or bs forms of Tbc1d20 were transiently transfected revealed a “loss-of-golgi” phenotype suggesting that overexpressed WT TBC1D20 strongly inhibits RAB1-mediated ER-Golgi transport, whereas overexpressed bs TBC1D20 did not disrupt the normal Golgi staining pattern. Additionally, BODIPY staining of WT and bs mEFs identified significantly larger and more abundant lipid droplets, implicating TBC1D20 as a negative regulator of lipid droplet formation, which may also be mediated through RAB1. In collaboration with other researchers, we also identified the first five human families containing mutations in Tbc1d20, with clinical presentations consistent with the Warburg Micro spectrum, a rare autosomal recessive disorder for which cataracts and hypogonadism are part of a severe clinical phenotype. (Abstract shortened by UMI.).