Oleg V. Bulgakov

Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Despite being the largest cytoskeleton, its structural composition has remained unknown. Here, we report a novel 220-kD protein, designated rootletin, found in the rootlets of ciliated cells. Recombinant rootletin forms detergent-insoluble filaments radiating from the centrioles and resembling rootlets found in vivo. An mAb widely used as a marker for vertebrate rootlets recognizes an epitope in rootletin. Rootletin has a globular head domain and a tail domain consisting of extended coiled-coil structures. Rootletin forms parallel in register homodimers and elongated higher order polymers mediated by the tail domain alone. The head domain may be required for targeting to the basal body and binding to a kinesin light chain. In retinal photoreceptors where rootlets appear particularly robust, rootlets extend from the basal bodies to the synaptic terminals and anchor ER membranes along their length. Our data indicate that rootlets are composed of homopolymeric rootletin protofilaments bundled into variably shaped thick filaments. Thus, rootletin is the long-sought structural component of the ciliary rootlet.

Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells

Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large (≈600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inner segment recess that wraps around the connecting cilia, corresponding to the periciliary ridge complex described for amphibian photoreceptors. In sensory hair cells of the cochlea, it is associated transiently with the hair bundles during postnatal development. Targeted disruption of the Ush2a gene in mice leads to progressive photoreceptor degeneration and a moderate but nonprogressive hearing impairment, mimicking the visual and hearing deficits in USH2A patients. These data suggest that usherin is required for the long-term maintenance of retinal photoreceptors and for the development of cochlear hair cells. We propose a model in which usherin in photoreceptors is tethered via its C terminus to the plasma membrane and its large extracellular domain projecting into the periciliary matrix, where they may interact with the connecting cilium to fulfill important structural or signaling roles.

Gene Therapy with a Promoter Targeting Both Rods and Cones Rescues Retinal Degeneration Caused by AIPL1 Mutations

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). Gene defects in AIPL1 cause a heterogeneous set of conditions ranging from Lebers congenital amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. In mice, null and hypomorphic alleles cause retinal degeneration similar to human LCA and RP, respectively. Thus these mouse models represent two ends of the disease spectrum associated with AIPL1 gene defects in humans. We evaluated whether adeno-associated virus (AAV)-mediated gene replacement therapy in these models could restore PDE biosynthesis in rods and cones and thereby improve photoreceptor survival. We validated the efficacy of human AIPL1 (isoform 1) replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene, which is active in both rods and cones. We found substantial and long-term rescue of the disease phenotype as a result of transgene expression. This is the first gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. We propose that the vector and construct design used in this study could serve as a prototype for a human clinical trial.

FKBP8 is a negative regulator of mouse sonic hedgehog signaling in neural tissues.

Sonic hedgehog (SHH) is a secreted morphogen that regulates the patterning and growth of many tissues in the developing mouse embryo, including the central nervous system (CNS). We show that a member of the FK506-binding protein family, FKBP8, is an essential antagonist of SHH signaling in CNS development. Loss of FKBP8 causes ectopic and ligand-independent activation of the Shh pathway, leading to expansion of ventral cell fates in the posterior neural tube and suppression of eye development. Although it is expressed broadly, FKBP8 is required to antagonize SHH signaling primarily in neural tissues, suggesting that hedgehog signal transduction is subject to cell-type specific modulation during mammalian development.

Replacement Gene Therapy with a Human RPGRIP1 Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

Increased choroidal neovascularization following laser induction in mice lacking lysyl oxidase-like 1.

PURPOSE Age-related degradation of the elastic lamina in Bruchs membrane may have a permissive effect on the growth of choroidal neovascularization (CNV). This study investigated the influence of defective elastic fiber maintenance in the development of laser-induced CNV. METHODS A mouse lacking lysyl oxidase-like (LOXL)-1, an enzyme essential for elastin polymerization, was studied. The morphologic characteristics of the elastic lamina within Bruchs membrane were examined in mutant and wild-type (WT) eyes. Laser-induced CNV was evaluated by fluorescein angiography and choroidal flat mounts. Immunohistochemistry for elastin was performed on the CNV lesions, and vascular endothelial growth factor (VEGF) levels were determined by ELISA. Soluble elastin and matrix metalloproteinase (MMPs) levels were also analyzed by immunoblotting. RESULTS The elastic lamina of Bruchs membrane in the LOXL1-deficient mice was fragmented and less continuous than in the WT controls. The mutant mice showed increased levels of soluble elastin peptides and reduced elastin polymer deposition in neovascular membranes. Significantly larger CNV with greater leakage on fluorescein angiography developed in mutant mice. VEGF levels in the RPE/choroid were higher in the knockout mice on days 7 and 14 after laser (P < 0.05). MT1-MMP (MMP14) was also elevated after laser in the LOXL1 mutant eyes compared to the WT controls. CONCLUSIONS These results show that a systemic defect in elastic fiber deposition affects Bruchs membrane integrity and leads to more aggressive CNV growth. The latter may be partially mediated by abnormal signaling from the accumulation of soluble elastin peptides.

Photoreceptor Rescue by an Abbreviated Human RPGR Gene in a Murine Model of X-linked Retinitis Pigmentosa

The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether adeno-associated virus-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314 codons, ‘short form’) or 1/3 (126 codons, ‘long form’) of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3.