Steps Towards Treating Genetic Deafness

By Jason Socrates Bardi

"Jack" and "Dianne" are hard working midwesterners, sweethearts since high school, loving parents, and both carriers of a recessive gene defect that they are not aware of. Jack and Diane have four children and their youngest, call him "Jake," has the misfortune to have inherited copies of the bad gene from both parents.

Despite the fact that his parents and older siblings are all generally healthy, Jake is born without the ability to hear. Hearing aids prove to be of no help, so Jake, his parents, and siblings all learn sign language.

Jake has other problems, too. He has trouble balancing and is a late walker—not taking his first steps until he is almost two years old. Throughout his childhood, he has to hold on to something solid when he sits down. Around age 10, the real trouble starts. Jake starts to have difficulty seeing at night, and by the time Jake is in his late teens, he is completely blind and no longer able to communicate.

Welcome to the world of Usher syndrome.

Even if the imaginary couple described above knew their son had the very real and devastating genetic disease Usher syndrome, there would not be much they could do. For there is no cure for Usher syndrome, the leading cause of deaf–blindness in the United States.

In his laboratory space overlooking the East Torrey Pines Mesa, Associate Professor Ulrich Mueller reflects on this problem and how it relates to his decision to bring his laboratory and research program here from the Friedrich Miescher Institute in Basel, Switzerland. He is one of the newest members of the Department of Cell Biology at The Scripps Research Institute (TSRI) and a member of TSRI's Institute for Childhood and Neglected Diseases (ICND).

The ICND was a good fit for him scientifically, he says, because it has a number of researchers who study questions related to nervous system development and function. This complements his own interest in Usher syndrome and topics at the intersection of neuroscience and genetics.

Mueller is also concerned with other human genetic diseases related to mechanosensory preception, and there are a vast number. "One in 800 children is born hearing impaired," he says, "And age-related hearing loss is also a big problem in society." In fact, nearly two thirds of all people over the age of 70 suffer from serious hearing loss.

Out in the laboratory, signs of his recent move are everywhere. Half-unpacked boxes; large equipment looking for a permanent home; a pan with paint and a roller brush in it. Still, the laboratory looks amazingly busy—a few students or postdocs can be seen filling shelves. Mueller and his laboratory can count the number of weeks they have been here on one hand, and they can't wait to get started.

"Next week, we start doing experiments," says Mueller.

Leading Cause of Deaf–Blindness

The experiments that Mueller and his laboratory will begin next week concern a number of questions related to Usher syndrome and the underlying biology of mechanosensory perception, a broad area encompassing not only hearing, but also balance, and a number of other bodily functions, such as blood pressure and gastrointestinal regulation. Hearing, balance, and these other activities are all regulated by receptors that sense our physical environment—sound waves or positional cues, for instance—and change physical signals into electrochemical ones.

Usher syndrome was first described by the pioneering German eye doctor Albrecht Von Graefe in 1858 and is named after a contemporary of Von Graefe, the British doctor Charles Usher, who believed that this condition was inherited, passing from parents to their children.

About 10 percent of children who are born deaf have a form of Usher syndrome, and it is the major cause of deaf–blindness. According to the National Institute on Deafness and Other Communication Disorders, which is one of the National Institutes of Health, more than half of the estimated 16,000 deaf-blind people in the United States are believed to have Usher syndrome.

Treatments for Usher syndrome tend towards the adaptive—hearing aids, cochlear implants, orientation and mobility training, auditory training, and Braille instruction. Perhaps the dearth of preventative therapies or cures is not surprising given that the syndrome is not well understood.

"We focus on [Usher syndrome] both to learn something about the disease but also about mechanosensory preception," says Mueller. "The process of mechanosensory preception is not at all understood."

Finding the Genes that Cause Deafness

What is understood is the basic anatomy of mechanosensory perception, hearing, and deafness.

It all starts in the inner ear. Therein resides an organ called the cochlea, which detects physical sound waves. Also in the inner ear is the vestibule, which is an organ that detects gravity and motion. The close proximity and the identical components of these two explains something about the tendency for hearing and balancing problems to be closely related.

When waves of sound—from a shattering window, for instance—hit a person's ear, they travel into the ear cavity and hit a group of "hair" cells that lie within the cochlea. These are the sensory cells that actually detect the sound with arrays of actin-rich, hair-like "stereocilia" projecting from their surface. These stereocilia are connected to each other and move as a bundle, and when they move, ion channels in them open, letting ions pass into the cells, change the polarization of the cells, and alter the release of neurotransmitters from the hair cells.

This change is monitored by sensory neurons and other support cells surrounding the hair cells, which then communicate electrical signals of their own to the brain, where neurons in the auditory association cortex can then fire and interpret the sound as breaking glass.

In Usher syndrome and other "sensory neuronal" diseases that cause deafness, the hair cells in the cochlea are unable to maintain the symmetric arrays of what are known as "stereocilia." Somehow the genetic defects cause the stereocilia to splay and degenerate instead of making bundles.

"These are not structural abnormalities of the bones," says Mueller, "but a disease that directly affects the morphology of the sensors."

But this basic picture falls far short of being useful for designing therapies because it lacks the identities and mechanisms of action of many of the receptors and other molecules that control these processes—the ion channels, for instance, have never been identified. Mueller estimates that there are more than 150 separate genetic loci involved in diseases that cause deafness, and only a fraction of the genes in these loci have actually been cloned.

Finding ways to treat disorders in mechanosensory perception without these details is like trying to describe a pantomime you watched in the dark.

The Components of Perception

A few of the molecular components involved in mechanosensory perception and implicated in deafness, however, have been identified—both by Mueller and in other laboratories.

And Mueller and his laboratory are looking to find more of these components by developing models of deafness, looking for important proteins, and setting up in vitro and in vivo systems to test the effects of those proteins on mechanosensory pathways. They would also like to find possible therapeutic targets among these components.

The odds of achieving this goal are helped by the fact that the field has lots of good in vivo models that copy the disease phenotype and can be used for finding the genes that cause the disease. "This is fortunate," says Mueller. "A lot of human diseases are so complex that you can't make decent models."

Many of the models of deafness have been around for years, and some of them have names like jerker, waltzer, and shaker, which reflect phenotypes arising from inner ear defects and balance problems. Several other models have been created by Mueller and his colleagues.

These models can be used to "positionally clone" the genes that are responsible for the phenotype. Positional cloning traditionally entails the use of classical genetic mapping methods to confine the location of the gene to a particular area in the genome, extensive sequencing of the region in question, and the performance of computer-aided searches through databases to find homology between sequences in that region and known genes.

This has been a fruitful approach so far, and to date mutations in at least 50 genes that code for everything from cell surface receptors to soluble cytosolic proteins have been cloned through these methods and were found to contribute to deafness. These include five genes that lead to different forms of Usher disease.

Recently, Mueller and his laboratory published a paper in the Proceedings of the National Academy of Sciences showing that two of these Usher genes act in a common pathway and in physical contact with one another.

One is a receptor protein cadherin 23. "We think that this receptor connects the stereocilia and is intimately engaged in the mechanical perception process by regulating the mechanical properties of the cell," says Mueller.

This is an important role, because in the inner ear, stereocilia appear in symmetric arrays and have to be connected to each other at the tips, sides, and ankles to move as one unit and properly detect sound waves. Cadherin 23 appears to be one of the molecules that makes these connections.

Significantly, cadherin 23 also contacts the protein harmonin—both are expressed in the stereocilia of hair cells. Mueller and his colleagues found that cadherin 23 and harmonin interact and bind to each other through particular "PDZ domains" in the harmonin proteins.

This interaction seems to be necessary for hearing, and when the two proteins do not bind to each other, the integrity of the stereocilia is compromised. Mutations in either the cadherin 23 receptor or the PDZ domain of the harmonin protein can lead to Usher syndrome. Instead of normal, symmetrically arrayed stereocilia, the mutations cause the stereocilia to be splayed and unable to properly detect sound waves.

Also interesting is the finding that Mueller and his laboratory made that the cadherin 23 receptor appears to be alternatively spliced in the ear and in the eye.

"We think that you express two different complexes in the different tissues," says Mueller. "One that is optimized for mechanical sensation, and the other one [we think] somehow has to do with maintaining the photoreceptor layer."

This may explain why defects in the gene encoding the receptor protein lead to deafness at birth but contribute to blindness that is only fully manifested later in life, when the mechanosensors in the eye reach maturity.

Integrins and Neuronal Development

Another part of Mueller's laboratory is devoted to looking at developing stem cells and the mechanics of differentiation and migration—two of the most important processes in neuronal development.

During fetal development a fountainhead of brain cells, hundreds of millions of them, are produced through proliferation of stem cells. But at some point, these same cells have to stop dividing, differentiate, and migrate to the position where they exert their function as specialized neuronal cells. Peripheral nerve cells also have to migrate to reach the area in the body where they exert their function. While migrating, these cells interact with other cells, which act as guideposts along the way.

"What we're trying to understand," he says, "is how a stem cell or a committed neuronal precursor decides to acquire a particular differentiated state and migrate to a particular position."

When a cell changes from proliferation to differentiation into a specialized cell, there are any number of gross and subtle changes in the expression of genes within that cell. Mueller and his laboratory look at expression profiles related to the differential state of the cells, trying to get at the molecular machines that regulate the switch from a proliferating cell to a migrating, differentiating neuron.

They are also interested in how the mutations in the genes they identify might contribute to pathology of central nervous system diseases. Once he finds genes that he thinks will be interesting to study further, he looks to perturb that gene and probe its function by knocking out genes expressed in the central nervous system through a sophisticated tissue-specific gene targeting technology.

One of the genes that the lab recently knocked out in the central nervous system (CNS) is the integrin subunit beta-1, which dimerizes with a number of different alpha subunits to form a functional integrin. Integrins are what cells use to cling to the extracellular matrix.

Knocking out the integrins in the entire organism is not possible because these adhesion molecules are necessary for maintaining the integrity of tissue. However, knocking them out specifically in the CNS creates a model with defects in the neuronal glial cells that resembles a set of human conditions known as lissencephaly, which comes from the Greek root for "smooth brain."

This model displays phenotypes that resemble those of a number of rare human diseases as well, such as Walker-Warburg Syndrome, muscle-eye-brain disease, and Baraitser-Winter syndrome.

All of these diseases have common features, including smoothness of the brain and defects in the brain's basal membrane. Patients with these diseases also suffer, to a certain degree, from retinal abnormalities, peripheral neuropahy, and muscular dystrophy. Similarly, patients with non-lissencephalous forms of muscular dystrophy sometimes have impaired mental function.

"What this all points to is a common molecular pathway for these different human diseases that involves interactions of cells with the extracellular matrix," says Mueller. "All these diseases are not so unrelated."

A Working Laboratory—Almost

Coming to TSRI and the ICND was a homecoming of sorts for Mueller. He spent the majority of his graduate years in the United States, working in the Lewis Thomas Laboratory at Princeton University, and completing his postdoctoral work at the University of California, San Francisco.

The move has not been simple. Setting up any new laboratory is difficult, but moving an existing laboratory from a European country to the United States offers its own unique challenges. Much of his time in the last year has been occupied working with the U.S. Department of Agriculture to get hundreds of serum samples, antibodies, and other biological compounds documented and approved for shipping and arranging visas for his group. Mueller arrived with three graduate students, one laboratory technician (now manager), and two postdoctoral fellows—a few of whom came a little early to start making arrangements.

Now, near the end of this long process, he is looking forward rather than back.

"March was the deadline I set to start to do experiments," says Mueller. "We're almost there—today we start doing little things, and next week, we're on the bench."



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Associate Professor Ulrich Mueller recently moved his laboratory and research program to TSRI. Photo by Jason Bardi.