Description of Research Expertise

Research Interests
Molecular Bases of Inherited Retinal Degenerations, Cell Biology of Photoreceptor Cells

Key words: Cilia, gene targeting, photoreceptor cells, macular degeneration, proteomic analysis, retinal degeneration, retinitis pigmentosa, RP1, RNA splicing

Description of Research
The overall goal of research in my lab is to help delineate the molecular bases of inherited retinal degenerations, so that improved therapies can be devised to prevent vision loss from these blinding disorders. Inherited retinal degenerations and dystrophies are major causes of blindness. These diseases are characterized by degeneration of the photoreceptor cells of the retina, resulting in loss of vision. There are no known cures for these diseases at present. The most common inherited form of blindness is retinitis pigmentosa (RP), a group of hereditary eye diseases characterized by gradual loss of peripheral visual field, and eventually loss of central vision in most cases. The most common cause of visual disability in the developed world is macular degeneration, in which central vision is lost. We are currently investigating the pathogenesis of several forms of RP and one form of macular degeneration, as described below.

We are also interested in the cell biology of the light sensitive photoreceptor cells of the retina. The outer segments of these cells are specialized sensory cilia, with stacks of membrane discs organized adjacent to an axoneme. We are interested in how these photoreceptor sensory cilia develop and are maintained, and how these processes are disrupted in disease.

Specific projects include:

RP1
Mutations in RP1 gene are a common cause of RP, but the function of the RP1 protein in vision, and how mutations in RP1 lead to photoreceptor cell death, are not understood. We are using biochemical and cell biologic techniques to study the function of the RP1 protein in vision. To date, we have found that RP1 is a photoreceptor-specific microtubule-associated protein (MAP) that is part of the axoneme of rod and cone photoreceptors. We have also used gene-targeting techniques to produce mice that express mutant RP1 in their retinas. In these mutant mice, the outer segment discs of rods and cones form, but fail to stack up into normal outer segments, indicating that RP1 may have a role in outer segment formation. We are now searching for proteins that interact with RP1 in order to further define its function, and study how the mutations lead to photoreceptor cell death. We are also starting to test potential therapies for RP1 disease.

Proteomics of Photoreceptor Sensory Cilia
The photoreceptor sensory cilium (PSC) is comprised of the outer segment and its cytoskeleton, including the ciliary rootlet, basal body and axoneme. Mutations in genes that encode PSC proteins are common causes of inherited retinal degenerations. We are using proteomic analyses to identify all of the proteins in PSCs. We are working to validate the PSC proteome experimentally, and investigate the function of selected proteins in PSC and maintenance.

RNA Splicing Factor Proteins and Retinitis Pigmentosa
Recently, 3 forms of RP have been found to be caused by mutations in ubiquitously expressed RNA splicing factors. Each of these splicing factors is a component of the spliceosome, the protein/RNA complex that is responsible for processing newly transcribed RNA into mature messenger RNA (mRNA). The identified genes encode pre-RNA processing factors (PRPF) 3, 8 and 31, which are defective in RP18, RP13 and RP11, respectively. It is not clear how mutations in such ubiquitously expressed proteins cause retina-specific disease. In order to study how mutations in these splicing factor proteins lead to blindness, we used gene targeting techniques to produce mice with mutations in Prpf3 and Prpf8. We are now investigating the effects of mutant Prpf3 and Prpf8 on RNA splicing in the retina and other tissues of these mice.

Inherited Macular Degeneration
Age-related macular degeneration (AMD) is one of the most common cause of vision loss in developed countries. The most characteristic clinical finding in the retinas of patients with AMD is drusen, or extracellular deposits of protein, lipid and debris that accumulate underneath the retinal pigment epithelium (RPE). At present, the etiology of drusen in AMD is not known, and there are only limited treatments are available to prevent the progression of AMD. In order to gain insight into the pathogenesis of AMD, we are studying an inherited form of macular degeneration called Doyne honeycomb retinal dystrophy (DHRD)/Malattia Leventinese (ML). Both DHRD and ML are caused by a single mutation, Arg-345 to Trp (R345W), in the EFEMP1 or Fibulin-3 gene. We have used gene targeting techniques to introduce this mutation into the Efemp1 gene of mice. We have found that the Efemp1-R345W knockin mice develop AMD-like deposits under their retinas, and are now studying the pathogenesis of these lesions.

Oligonucleotide-directed Gene Targeting
In addition to our work on retinal degenerations, we are also interested in developing new methods for producing mice with specific single-base mutations to use as models of inherited diseases. We are using small oligonucleotides to introduce mutations into the DNA of mouse embryonic stem (ES) cells. To date, we have demonstrated that our oligonucleotides can produce single-base changes in reporter genes in mouse ES cells. We are now working toward using oligonucleotide-directed gene targeting to modify endogenous genes in ES cells. Mice generated from the targeted ES cells could be used to study the pathogenesis of inherited retinal degenerations, or other inherited diseases.

Rotation Projects for 2005-2006
1. RP1 protein function and therapies
2. Proteomic analyses of photoreceptor outer segments
3. RNA splicing defects in Prpf3 and Prpf8 mutant mice
4. Characterization of AMD-like deposits in Efemp1 mutant mice

Lab personnel:
Li Fu, M.D., Ph.D. – Research Project Manager
John Graziotto – Graduate student, Neuroscience
Qin Liu, M.D., Ph.D. – Research Assistant Professor
Hamideh Moayedpardazi – Undergraduate
Brian Murphy – Graduate student, IME
Alex Saveliev – Research specialist
Jonathan Weiner – Undergraduate
Qi Zhang, Ph.D. – Senior Research Investigator

Description of Clinical Expertise

Pediatric Ophthalmology

Description of Other Expertise

Techniques for gene targeting in mouse ES cells