Scientists at Oregon State University (OSU) say they have found a new piece of the puzzle in the quest to use gene therapy to enable people born deaf to hear. The work centers around a large gene responsible for an inner-ear protein, otoferlin. Mutations in otoferlin are linked to severe congenital hearing loss, a common type of deafness in which patients can hear almost nothing.
“For a long time otoferlin seemed to be a one-trick pony of a protein,” said Colin Johnson, PhD, associate professor of biochemistry and biophysics in the OSU College of Science. “A lot of genes will find various things to do, but the otoferlin gene had appeared only to have one purpose and that was to encode sound in the sensory hair cells in the inner ear. Small mutations in otoferlin render people profoundly deaf.”
In its regular form, the otoferlin gene is too big to package into a delivery vehicle for molecular therapy, so Johnson’s team is looking at using a truncated version instead.
Research led by graduate student Aayushi Manchanda showed the shortened version needs to include a part of the gene known as the transmembrane domain, and one of the reasons for that was unexpected: Without the transmembrane domain, the sensory cells were slow to mature.
“That was surprising since otoferlin was known to help encode hearing information but had not been thought to be involved in sensory cell development,” Johnson said.
The study (“Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking”) appears in Molecular Biology of the Cell.
“Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish,” wrote the investigators.
“At the early developmental timepoint of 72 hours post-fertilization (hpf) low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after upregulation of mutant otoferlin.
“Analysis of membrane-reconstituted otoferlin measurements suggest a function for the transmembrane domain in liposome docking. We conclude that that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.”
Scientists in Johnson’s lab have been working for years with the otoferlin molecule and in 2017 they identified a truncated form of the gene that can function in the encoding of sound.
To test whether the transmembrane domain of otoferlin needed to be part of the shortened version of the gene, Manchanda introduced a mutation that truncated the transmembrane domain in zebrafish.
“The transmembrane domain tethers otoferlin to the cell membrane and intracellular vesicles but it was not clear if this was essential and had to be included in a shortened form of otoferlin,” Manchanda said. “We found that the loss of the transmembrane domain results in the sensory hair cells producing less otoferlin as well as deficits in hair cell activity. The mutation also caused a delay in the maturation of the sensory cells, which was a surprise. Overall the results argue that the transmembrane domain must be included in any gene therapy construct.”
At the molecular level, Manchanda found that a lack of transmembrane domain led to otoferlin failing to properly link the synaptic vesicles filled with neurotransmitter to the cell membrane, causing less neurotransmitter to be released.
“Our study suggests otoferlin’s ability to tether the vesicles to the cell membrane is a key mechanistic step for neurotransmitter release during the encoding of sound,” noted Manchanda.