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Commentary Free access | 10.1172/JCI36515
1Department of Ophthalmology and Visual Sciences and 2Department of Physiology, University of Kentucky, Lexington, Kentucky, USA.
Address correspondence to: Jayakrishna Ambati, Department of Ophthalmology and Visual Sciences, E300 Kentucky Clinic, 740 S. Limestone Street, Lexington, Kentucky 40536-0284, USA. Phone: (859) 323-0686; Fax: (859) 323-1122; E-mail: jamba2@email.uky.edu.
Find articles by Kleinman, M. in: JCI | PubMed | Google Scholar
1Department of Ophthalmology and Visual Sciences and 2Department of Physiology, University of Kentucky, Lexington, Kentucky, USA.
Address correspondence to: Jayakrishna Ambati, Department of Ophthalmology and Visual Sciences, E300 Kentucky Clinic, 740 S. Limestone Street, Lexington, Kentucky 40536-0284, USA. Phone: (859) 323-0686; Fax: (859) 323-1122; E-mail: jamba2@email.uky.edu.
Find articles by Ambati, J. in: JCI | PubMed | Google Scholar
Published July 24, 2008 - More info
Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.
Zhenglin Yang, Yali Chen, Concepcion Lillo, Jeremy Chien, Zhengya Yu, Michel Michaelides, Martin Klein, Kim A. Howes, Yang Li, Yuuki Kaminoh, Haoyu Chen, Chao Zhao, Yuhong Chen, Youssef Tawfik Al-Sheikh, Goutam Karan, Denis Corbeil, Pascal Escher, Shin Kamaya, Chunmei Li, Samantha Johnson, Jeanne M. Frederick, Yu Zhao, Changguan Wang, D. Joshua Cameron, Wieland B. Huttner, Daniel F. Schorderet, Frances L. Munier, Anthony T. Moore, David G. Birch, Wolfgang Baehr, David M. Hunt, David S. Williams, Kang Zhang
Although age-related macular degeneration is the most prevalent macular disease in the world, numerous discoveries regarding the molecular bases of vision have been made through genetic association studies of rare inherited maculopathies. In this issue of the JCI, Yang et al. present a functional genetics study that identifies a role for prominin 1 (PROM1), best known as a stem cell and/or progenitor cell marker, in the biogenesis of retinal photoreceptor disk arrays (see the related article beginning on page 2908). This study supports an established model in which disk morphogenesis occurs through membrane evagination and extends other recent studies assigning PROM1 important functions outside of the stem cell niche.
More than 50 years ago, the first ultrastructural evidence of photoreceptor disk organization was published by noted electron microscopist Fritiof Sjöstrand (1). Subsequent studies provided more detailed characterizations of the evolutionarily conserved arrangement of rod and cone photoreceptors into inner and outer segments within Bilateria (2). It is in this outer segment region that thousands of rhodopsin-containing bilayered disks form an array of photovoltaic cells that transmit visual stimuli to the neural retinal components. Without the organized development and maintenance of these precious subcellular elements, the eye cannot fulfill its raison d’être.
Many congenital and acquired diseases that result in vision loss are caused by photoreceptor degeneration. The most widely studied of these pathologies is age-related macular degeneration (3), an epidemic in the developed world affecting approximately 30–50 million people, rivaling the prevalence of cancer (4). However, the study of other, more rare hereditary macular diseases has also yielded fundamental knowledge that has greatly advanced our understanding of the molecular bases of vision. Historically, many of these major studies were published in 2 phases: the genetic association data was followed by insights into the functional implications of an identified polymorphism obtained via the use of transgenically engineered mice. In this issue of the JCI, Yang et al. give us the best of both worlds by presenting a combined functional genetics investigation of the critical nature of prominin 1 (PROM1; also known as CD133 and AC133) expression during photoreceptor disk morphogenesis that provides essential insight into the molecular programming of disk formation and the ever-expanding roles for PROM1 (5).
PROM1 is still best known for its original use as a human stem cell–specific marker (6), yet its known biological functions continue to reach far beyond this role. The protein is constructed of 5 transmembrane domains, 2 large extracellular loops containing 8 N-linked glycosylation sites, and a cytoplasmic tail. Variable glycosylation of these extracellular loops may account for the monoclonal antibody specificity for certain tissue types and circulating stem cells. Contemporaneous with the characterization of AC133 for hematopoietic cell lineage analysis, another group reported the discovery of a mouse protein, termed PROM1, found to be expressed on specific embryonic and adult epithelia and localized to plasma membrane protrusions (7). Although it was quickly realized in an exchange of public letters by the 2 laboratories that the human stem cell marker was the likely homolog of mouse PROM1, with more than 60% sequence overlap, an entire body of literature emerged in which the antigen was used to identify specific cell populations. In a recentJCI article, previously unchallenged claims that PROM1 was a marker of tumor-initiating metastatic colon cancer cells were rebutted in a study that demonstrated the initiation of colon cancer tumors in xenografts by PROM1-negative cells (8). Thus, it appears that PROM1 is not as lineage specific or functionally determined as it once was purported to be.
There is mounting evidence that PROM1 is critical to the organization of photoreceptor disks. In 2000, a group that included members from the team that initially described mouse PROM1 found a genetic association between a human PROM1 frameshift mutation and a form of autosomal-recessive retinal degeneration in a small Indian pedigree (9). This polymorphism resulted in premature termination of the protein, which prohibited it from reaching the cell surface. In these studies, PROM1 was found to localize to the base of the outer segment of murine rod photoreceptors, where disk biogenesis occurs. Yet the precise implications of this focused expression remained undefined. Yang et al. now provide us with critical information regarding the functionality of PROM1 in photoreceptor disk formation through an integrated approach that elegantly couples genetic association data with an in vivo animal study (5).
After identifying 2 pedigrees with different forms of inherited autosomal-dominant macular degeneration, the team mapped the 2 phenotypes to a region on chromosome 4p (5). In further genetic screening analyses of this region, a missense mutation was found in the coding sequence of the PROM1 gene that resulted in the replacement of arginine with cysteine at amino acid position 373 (R373C). Importantly, mutation analyses of a third pedigree with an autosomal-dominant cone-rod dystrophy also revealed the R373C polymorphism, demonstrating that the PROM1 mutation is linked to 3 forms of dominant macular degeneration in humans.
To shed light on the biological effects of R373C in vivo, transgenic mice were engineered containing either the wild-type or the R373C mutated human PROM1 gene under the control of the rhodopsin promoter, thereby localizing expression to the rod photoreceptors (5). In mice with the mutant PROM1, serial retinal imaging exhibited findings consistent with those found in humans. Progressive photoreceptor degeneration was evident both in histological analyses and in analysis by electroretinography, a functional modality commonly used to quantify retinal response to light. The electron microscopy studies revealed a much more significant finding from this paper, that PROM1 appears to direct the organization of photoreceptor disks. Mice expressing the mutant PROM1 gene had malpositioned and overgrown disk membranes. As has been proposed elsewhere (10), PROM1 may be responsible for proper nascent disk alignment into bilayers (Figure 1). This mechanism is suggested by the presence of extracellular, leucine-like zipper motifs and the potential for PROM1 dimerization to link plasma membrane protrusions. These observations open a new avenue for investigation in the search for a molecular explanation of photoreceptor disk morphogenesis, a longstanding question in photoreceptor cell biology in particular and developmental biology in general.
Retinal rod photoreceptor disk assembly requires PROM1. The rod photoreceptor cell consists of an outer segment containing an array of rhodopsin-loaded disks, a myoid region containing mitochondria, nucleus, and other organelles, and a synapse region that connects to the neural retinal network to transmit visual stimuli. PROM1 normally localizes to the nascent disk membranes, but in the case of the existence of an R373C mutation, the protein remains in the myoid region. In their study in this issue of the JCI, Yang et al. show that PROM1 interacts with protocadherin 21 (PCDH21) and actin filaments to regulate disk morphogenesis and subsequent maturation from evaginating nascent disk membranes (5).
The study by Yang et al. (5) is a timely contribution to the field of retinal cell biology because it supports a hypothesis on photoreceptor disk formation that gained acceptance over the past 4 decades but has recently been challenged. Critical data presented here and elsewhere suggest that PROM1 is localized to the base of the outer segment and that without its functional presence, erroneous disk formation occurs. These findings align with a model of disk biogenesis wherein the outer segment base serves as the membrane source for disk renewal, a concept that has been supported by numerous other investigators. In 1964, a student from Sjöstrand’s lab published an electron microscopy study of amphibian retina showing evaginations of the cell membrane of the photoreceptor outer segment (11). Several scientific giants went on to pioneer this field, including Richard Young, who rigorously studied the ultrastructure of photoreceptor elements in monkeys (12), and Roy Steinberg, who proposed an open model of disk biogenesis consisting of 2 membrane growth phases: evagination of the ciliary plasma membrane and formation of the disk rim (13). Further molecular work began to unravel the kinetics of disk formation and shedding (14, 15) as well as the protein interactions required for disk rim formation during photoreceptor membrane evagination (16). Now Yang et al. make another considerable stride by demonstrating that interactions among PROM1, protocadherin 21, and cytoskeletal actin regulate the outgrowth of evaginations of the plasma membrane of photoreceptor cilia, further supporting the open model of disk membrane formation (5).
In a significant departure from this hypothesis, a recent paper in Cell presented data suggesting that disks grow by fusion between opsin-containing vesicles and nascent disk membranes (17). Yang et al. argue strongly against this hypothesis with molecular and mutant phenotypic evidence that dually supports the earlier model (5). The fusion model has generated much skepticism because it is contradicted by an enormous cache of data showing nascent membrane formation at the base of the outer segment and the currently accepted open disk model of cone photoreceptor ultrastructure. The controversial data advancing the fusion model may be due to the choice of fixation agent, acrolein, which can significantly alter photoreceptor membranes (18) as well as 3-dimensional skew as a result of section orientation. Regardless of the potential shortfalls, in this era of advancing molecular imaging, a conclusive resolution should be attainable. To date, freeze fracture studies of photoreceptor membranes have not provided a definitive ruling regarding the validity of the fusion model (19), but to our knowledge, a rigorous electron microscopic analysis of cryopreserved eyes has not been reported. Such a study visualizing the unperturbed membranes of the photoreceptor disk might unequivocally capture the functional morphology. Emerging 3-dimensional electron microscopy technologies could be leveraged to address this question. An alternative approach may be to use probes that can target the photoreceptor membrane and be secondarily labeled for imaging by electron microscopy or other new nanometer resolution systems. One such probe is a recently described actin-binding oligopeptide capable of nondestructive live visualization of cytoskeletal dynamics in vivo (20).
In conclusion, the discovery of PROM1-associated macular degenerations simultaneously reveals an important molecular mechanism for photoreceptor disk formation and widens the biological ambit of PROM1 (5). In nature’s mind, it would be woefully inefficient to create a unifunctional molecule. The growing recognition of PROM1’s functional diversity attests to this notion and invites the continued investigation of its physiologic and clinical importance. It is also critical to take into account the gamut of biological roles for PROM1, especially given that targeted therapeutics are currently being developed for the treatment of some PROM1-expressing cancers (21).
The authors’ research is generously supported by the Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Research Endowed Chair, a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Research to Prevent Blindness Lew R. Wassermann Merit and Physician Scientist Awards, the American Health Assistance Foundation, a University of Kentucky University Research Professorship, and NIH grants EY015422, EY018350, and EY18836 (to J. Ambati) as well as by the International Retinal Research Foundation Charles D. Kelman Award (to M.E. Kleinman).
Address correspondence to: Jayakrishna Ambati, Department of Ophthalmology and Visual Sciences, E300 Kentucky Clinic, 740 S. Limestone Street, Lexington, Kentucky 40536-0284, USA. Phone: (859) 323-0686; Fax: (859) 323-1122; E-mail: jamba2@email.uky.edu.
Nonstandard abbreviations used: PROM1, prominin 1.
Conflict of interest: The authors have declared that no conflict of interest exists.
Reference information: J. Clin. Invest.doi:10.1172/JCI36515.
See the related article at Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.