Neuronal Retinal Degeneration: Photoreceptor Polarity and Vectorial Protein Transport
- Characterization of Retinitis Pigmentosa Rhodopsin Mutations
- Vectorial Trafficking and Polarity in the Rod Photoreceptor
- Cytoskeletal Specialization of RPE and Its Relationship to Retinal Diseases
Characterization of Retinitis Pigmentosa Rhodopsin Mutations
Retinitis pigmentosa (RP) is one of the most common hereditary retinal dystrophies, affecting approximately 1 in 4,000 people in all populations examined. RP typically begins with the loss of rod cell function, causing night blindness and loss of peripheral vision. Central vision, mediated by foveal cone cells, is also affected in the later course of the disease. The severity and onset of retinal phenotypes varies greatly depending on the mutation. To date, nearly 100 mutations of the gene encoding rhodopsin (the visual pigment of rod photoreceptor) have been identified in patients with autosomal dominant RP.
Class I
Class II
Class III
We have characterized and classified mutant rhodopsins based on their biochemical and cellular defects (i.e., protein folding, chromophore binding, G-protein coupling/activation, subcellular localization). Class I mutations are concentrated in the cytoplasmic C-terminal tail of rhodopsin. These mutants resemble wild type rhodopsin in the aspect of phototransduction. However, transgenic animal studies revealed that several class I mutant rhodopsins have defects in their polarized outer segment distribution in photoreceptors. Class II mutations are found throughout the entire coding sequence of rhodopsin. Class II mutants are defective in chromophore assembly. They are either completely or partially retained in the endoplasmic reticulum and fail to reach the cell surface. Class III mutations are only found at the Arg135 residue. Our data show that Arg135 mutants are constitutively hyperphosphorylated in the absence of chromophore and interact with visual arrestin with high affinity. The rhodopsin-arrestin complexes profoundly attenuate both the organization and function of the endocytic compartments. We propose that impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations. It is interesting to note that patients with an Arg135 mutation usually experience much faster progression to blindness than patients carrying most other rhodopsin mutations.
Patients carrying rhodopsin mutations account for ~25% of autosomal dominant RP cases. Correct diagnosis of the molecular defect of each class of mutant may lead to an effective treatment for a specific type of RP. In addition to our effort in dissecting the biochemical and cellular defects of various RP mutant rhodopsins, we aim to understand how the impairment of rhodopsin's function leads to rod cell death and then to subsequent cone cell death. A fuller understanding of these questions may provide a chance to intercept these processes and, hence, delay the progression of the disease and preserve vision.
References
Sung, et al., Proc. Natl. Acad. Sci. USA 88: 6481-6485 (1991).
Sung, et al., Proc. Natl. Acad. Sci. USA 88: 8840-8844 (1991).
Sung, et al., J. Biol. Chem. 268: 26645-26649 (1993).
Portera-Cailliau, et al., Proc. Natl. Acad. Sci. USA 91: 974-978 (1994).
Sung, et al., J. Neurosci. 14: 5818-5833 (1994).
Sung and Tai. International Review of Cytology 195: 215-267 (2000).
Sung and Chuang. In Cell Biology and Related Disease of the Outer Retina (Williams, D. eds). World Scientific Co. (2004).
Chuang, et al., J. Clin. Invest. 114:131-140 (2004).
Vectorial Trafficking and Polarity in the Rod Photoreceptor
The rod photoreceptor is a light-responsive neuron that is highly polarized in both morphology and function. The vertebrate rod has evolved to have a specialized light sensing organelle called the outer segment (OS). The OS consists of a plasma membrane that encloses a stack of ~1,000 closely spaced discs and a connecting axonemal segment that serves as a bridge to the cell body. A remarkable feature of the photoreceptor is that the OS are continuously and rapidly renewed throughout the lifetime of the animal. The distal tip of the OS is regularly shed and phagocytosed by the adjacent cells of the retinal pigment epithelium (RPE), while at the base of the OS, new disks are assembled from new proteins and lipids that were synthesized in the IS.
In rods, the visual pigment rhodopsin is highly concentrated in the OS, where phototransduction occurs. This distribution of rhodopsin may represent the most extreme example of polarized protein distribution in all systems examined so far. Impaired OS targeting of rhodopsin has been linked with several retinal degenerative diseases. The long-term goal of this lab is to unravel the molecular details underlying the translocation steps that deliver rhodopsin (and other photoreceptor proteins) from the site where it is synthesized to where it functions, how the transport is regulated, and how defective rhodopsin transport leads to the pathogenesis of RP. Answers to these questions will further our insight into the fundamental process of the vectorial transport of photoreceptor proteins, and the genesis and maintenance of the polarity of visual cells. In addition, these studies may have important implications for future rational therapies for the diseased retina.
Toward this aim, our lab has identified proteins that interact with rhodopsin's OS targeting signal (i.e., its cytoplasmic tail) by yeast two-hybrid screening. The physiological relevance of these protein-protein interactions is then followed by an array of biochemical, cell biology, histological, and animal studies. Finally, the in vivo retinal transfection technique, pioneered by Takahiko Matsuda and Connie Cepko at Harvard, has been integrated in these analyses to facilitate our studies of mammalian photoreceptors in vivo. Some of these studies have shown that dynein- and microtubule-based transport involves the post-Golgi vesicular transport of rhodopsin in the IS and that the docking of rhodopsin vesicles on microtubules is mediated by the specific protein interaction between the rhodopsin C-terminus and the dynein subunit Tctex-1.
References
Sung et al., J. Neurosci. 14: 5818-5833 (1994).
Tai et al.,. J. Biol. Chem. 273: 19639-19649 (1998).
Chuang and Sung. J. Cell Biol. 142: 1245-1256 (1998).
Tai et al., Cell 97: 877-887 (1999).
Tai et al., J. Cell Biol. 153:1499-1509 (2001).
Cytoskeletal Specialization of RPE and Its Relationship to Retinal Diseases
The retinal pigment epithelium (RPE) is a multifunctional and indispensable component of the vertebrate retina. Some of the features of RPE that are essential for its function are: asymmetry, specialized membrane structures, and membrane motility. All of these features rely on a highly ordered cytoskeleton. At present, little is known about the machinery and the molecular mechanism regulating cytoskeleton-mediated RPE functions. RPE morphology undergoes profound changes during aging and in some types of retinal degeneration (e.g., age-related macular degeneration and proliferative vitreoretinopathy). These phenotypes are often accompanied by disorganization and death of photoreceptors. However, the chronology of the morphological changes in the aged and/or diseased RPE cells has not been pieced together. The causal relationship between the morphological changes and functional impairment of RPE/neural retina are also not fully understood.
Our lab seeks answers to these questions by focusing on the functional importance of the actin cytoskeleton and its associated proteins in RPE during morphogenesis and aging. We combine genomics, mouse genetic manipulations, and imaging techniques to tackle these questions.