Usher syndrome, part III: the plot thickens

Guest Blogger Danio:

The time has come to delve into the retinal component of Usher syndrome. In Part II, I briefly described the results of protein localization studies, in which most members of the Usher cohort were found at the connecting cilium of the photoreceptor and at the photoreceptor synapse. The following diagram summarizes these findings:

Usher protein localization in photoreceptor cells. From Reiners, et al. 2006

So, as we saw in the ear, proteins with the equipment for physically interacting with one another are gathering in specific places, and thus multi-protein complexes are likely being formed at these locations. The cluster of Usher proteins around the connecting cilium has been the focus of most of the current retinal studies, and to understand the potential importance of an Usher complex at that subcellular location we must address the importance of the connecting cilium itself.

Recall the structure of the photoreceptor cell described in Part I. The inner segment, just above the nucleus, contains all the standard-issue cell operating equipment--organelles required for producing protein, degrading cellular waste products and performing various other metabolic functions. The outer segment contains the intricately folded membrane discs with which light sensitive molecules are associated. Between these two cellular compartments lies the connecting cilium, which grows out of the inner segment, extends up into the outer segment, and is surrounded by a structure known as the periciliary ridge, which encircles the cilium like a little cuff. The cilium serves as a functional connection between the inner and outer segments, as well as a structural one. Proteins and other cellular materials synthesized in the inner segment need to get to the outer segment in order to perform their particular jobs up there, and materials that are no longer needed in the outer segment need to be carried away and dealt with in the inner segment. The connecting cilium acts as a transport system to which motor proteins can anchor and pull their molecular cargo up or down as needed.

The localization studies of the Usher proteins reveal that they are in the vicinity of the connecting cilium, but a closer look at this region of the cell shows that they are specifically either in the periciliary ridge--the 'cuff'--or the space between the periciliary ridge and the connecting cilium:

Schematic longitudinal (B) and cross (C) sections of the ciliary/periciliary region of a mammalian photoreceptor cell showing the localization and proposed activity of some interacting Usher proteins. From Märker, et al. 2008

Thus, the model for usher protein function in the retina is that these complexes somehow assist in the cilium-based transport system. Here's where things get a bit murky, though. Remember that, unlike the congenital nature of the ear problems, the retinal symptoms don't manifest until much later, in childhood or adolescence,. Furthermore, they progress quite slowly, well into the third decade of life in most cases. How can the same defective proteins that cause such significant developmental problems in the hair cells not cause early problems with retinal cell function as well?

The congenital deafness in human patients and mouse models of the disease, and the defects in stereocilia formation seen in the Usher mice are nicely explained by the model of protein interaction and function in the developing hair cells, discussed in Part II. The retinal cells, however, survive development and, apparently, function normally until they begin to degenerate. I say 'apparently' because the 'pre-death' state of the photoreceptors has been difficult to observe thus far. In human patients, the first sign of a problem occurs when the hearing-impaired child or teenager begins to experience night blindness due to a loss of rod photoreceptors in the periphery of the eye. By the time this can be detected clinically, the degeneration is already well underway, and although the progressive vision loss is gradual, ophthalmic examinations haven't yet been able to identify any problems that precede the cell death.

At this point you might well ask what clues the Usher mice, which proved so valuable in adding to our understanding of the disease progression in the ear, can tell us about the events leading up to retinal degeneration. To our great consternation, most of the Usher mice do not undergo retinal degeneration at all! The Usher mutant lines have all been examined expectantly until the end of their natural lives (around 2 years) and most do not exhibit any abnormality in their retinas. The exceptions to this are older mice with mutations in the cadherin 23 (ush1d) gene, which show a slight reduction in visual function older ages, and myo7a mutant mice, which exhibit a fairly distinct defect in protein trafficking, lending support to the model of usher proteins at the connecting cilium as described above. Neither line shows any retinal degeneration, however.

Several theories have been put forth to explain this discrepancy between the mouse and human forms of the disease. One possibility is that mice, being nocturnal animals and usually raised in low-light laboratory conditions, may not endure the bright light exposure that human retinas must withstand. Another explanation may lie in the slow, progressive nature of the human disease and the relatively short life cycle of the mouse--perhaps two years just isn't long enough for the retinal defects to manifest in the mouse retina. A third theory centers on the fact that all of the known Usher proteins actually exist in multiple isoforms. The genetic code that specifies each of these proteins can be cut and spliced in a few different ways. The exact roles of the different isoforms of every gene aren't yet clear, but some of them do appear to be more important in the ear than in the eye. It's possible that the mutations in mouse Usher genes that give rise to such a strong ear phenotypes don't affect the part of the protein that's important for retinal cell function, and thus the mouse is spared the vision loss that characterizes the human disease. In further support of this latter theory is that fact that many of the Usher syndrome genes are also linked to non-syndromic deafness in humans--hearing loss without associated blindness.

None of the above theories are mutually exclusive, and it may turn out to be a combination of genetics, environment and life-span that has limited the retinal phenotype of the Usher mutant mice. Encouragingly, significant progress has been made through the use of genetically engineered mice, in which an Usher protein is removed completely (see knockout mice for more on this technique) or, alternatively, a targeted mutation is introduced into a particular Usher gene that renders the encoded protein non-functional. Thus far, these genetically modified mice show late-onset retinal degeneration, usually detectable at around 20 months of age. Exhibiting the full range of Usher syndrome symptoms, these new mouse models will provide hitherto unavailable opportunities for exploring the still mysterious function of the Usher proteins in the retina.

Hopefully, the above summary has illustrated that there are still a great many unanswered questions surrounding the pathophysiology of Usher syndrome, particularly with respect to the timing and progression of the retinal defects. To complement the data being collected from the current mouse models, I have chosen to investigate the function of various Usher proteins in the zebrafish. There are some differences in the retinal anatomy of zebrafish and humans, but basic cell structure and function is conserved between the two species. Additionally, there are some similarities that make zebrafish an especially appealing organism for this type of study, including the fact that fish are diurnal animals with rich color vision--even better than humans, in fact, as they can see light in the ultraviolet range of the spectrum. Other advantages to using zebrafish are related to their development. Zebrafish embryos undergo fertilization and development outside the mother's body, and usually several hundred embryos are produced from a single mating. They develop rapidly and are able to swim, see and hear just a few days after fertilization. Thus, I am able to begin conducting tests on their visual function within the first week of development and obtain results quite rapidly compared to the work being done in mammals.

I am in the process of making Usher mutant zebrafish lines using a new technique for targeted mutagenesis. In the meantime, I am able to use established techniques to study the consequences of depleting Usher proteins in larval zebrafish. Although most of these data have yet to be published, I can report that, along with balance and hearing defects, I am seeing problems with retinal cell function, and I am able to detect photoreceptor death in young fish in which the various Usher proteins have been disabled. Once the starting point of the cell death is pinned down for each of the genes, I examine various aspects of retinal cell structure and function prior to that time point to see if I can detect any abnormalities. Manuscripts are in preparation, so stay tuned.

Understanding the molecular events that precede the death of these cells will be crucial in identifying ways to improve diagnosis and treatment of Usher syndrome. In the conclusion of the Usher story, and of my Guest Blogging stint, I'll discuss current clinical practices for managing Usher syndrome, and the direction of the research efforts designed to enhance these treatments.

Figure credits: 1. Reiners J, et al. 2006Experimental Eye Research Volume 83, 97-119.
2. Märker T, et al. 2008 Human Molecular Genetics Volume 18, 71-86