Anyone who has ever planted something from seed knows it takes time for the resulting plant to bear fruit. AHRF’s seed grants take time to bear fruit, too.
In 2018, AHRF awarded a grant to Anna Lysakowski, professor in the Department of Anatomy & Cell Biology at the University of Illinois at Chicago (UIC) and co-PI Jose Antonio Lopez-Escamez, MD, PhD, then at Granada University in Spain (now at the University of Sydney), for a study addressing familial Meniere’s disease (MD). Although reports vary, the percentage of Meniere’s disease that is familial – that is, where there is a family history of the disorder – is estimated to be between 5-15% of all Meniere’s cases.
With results in hand, they and their colleagues pursued a larger grant from the National Institutes of Health (NIH). Study results were published in the August 2023 issue of European Journal of Neuroscience.
Here, Dr. Lysakowski explains where the AHRF-funded study led.
1) Why did you focus on familial Meniere’s disease (MD)?
My Spanish colleague, Jose Antonio Lopez-Escamez, MD, PhD, is a world’s expert on familial MD. He has discovered 8-9 new genes that are mutated with single nucleotide variations (SNPs) in MD patients and not in their unaffected siblings. I’d previously published two papers with him where my lab localized three of these gene products (proteins) in the inner ear, with immunohistochemical methods. The two genes, FAM136a and DTNA, had been found to be mutated in three generations of the same family, so it was unclear if they were each responsible for a different aspect of the MD. Meniere’s is more of a syndrome, like Down’s Syndrome (meaning that there are multiple components to the disease, balance disorder, progressive hearing loss, tinnitus, nausea, aural fullness, sudden vertigo or falling attacks), so there was likely not just one cause or mechanism involved.
2) What was the aim of the study?
So we had localized the proteins in the inner ear (first step, because it wouldn’t make any sense if these proteins were in the stomach instead of the inner ear), but we didn’t know if they actually caused vestibular or hearing problems. Hence, we looked for mouse models in which two of these specific genes were knocked out and tested them for vestibular and hearing problems.
The way we tested the mice in this study was to place them on a slowly rotating rod and then slowly speed up the rotations until they fell off (a short distance to the bottom of the enclosure), in a behavioral test called the RotaRod test. We compared wild type (WT) mice, those who didn’t have the gene knocked out, with the knockout (KO) mice, to see how long it took them to fall, the so-called “Latency to fall”. We also tested their hearing, in a somewhat crude fashion, by using a clicker to elicit a so-called “acoustic startle reflex”, which is a little jump when they heard the click. If they didn’t jump, then they didn’t hear the noise, so they were deaf.
3) What results did you get, and what do they mean?
We tested the same mice monthly, both males and females, once a month for 24 months (the mouse equivalent of a human 70 years old) to attempt to distinguish the effects of vestibular and hearing loss in the KO mice compared to normal aging.
The results were that sex, age, and genotype were significantly correlated with shorter “Latencies to Fall” for male DTNA KO mice, while only age was a significant factor for FAM136a KO mice. FAM136a KO mice, on the other hand, lost their hearing months before WTs (9–11 months vs. 15–20 months).
So, we concluded that the vestibular loss may be due to the DTNA mutation and the hearing loss to the FAM136a mutation. DTNA is found at synapses, the connections between hair cells and their nerve endings, and FAM136a is a mitochondrial protein of unknown function (there are lots of mitochondria in both hair cells and their nerve endings). So the two genes were responsible for different aspects of the disease.
4) How can these results shed light on MD in general?
These results highlight the idea that MD has multifactorial causes. Familial MD, as far as we know now, is responsible for 5-15% of MD cases (about the same proportion of the population as Huntington’s Disease, with the higher numbers being found in Asian populations). And it is a disease that usually manifests in middle age, so it’s not as if children get it, but rather adults in the prime of their working life, thus the economic affects can be devastating. So in addition to the genetic aspects, there are also autoimmune causes, MD cases related to vestibular migraine, MD cases related to stroke, and MD cases caused by endolymphatic hydros (excess fluid accumulation in the tight inner ear spaces). Some cases can be treated by giving the patients diuretics, but finding genetic causes raises the hope of gene therapy as a cure.
5) What could follow from these results? What’s next to investigate?
As mentioned above, gene therapy is a possible treatment, once we know the possible genetic causes.
What I would like to investigate is other genetic and/or sporadic causes. There are sporadic cases that may have genetic origins, for example, the gene OTOG (otogelin), a known hearing loss gene, has been implicated in familial MD as well. We wanted to study OTOG KO mice in the same manner, to characterize the disorder in this genetic mutation.
But other avenues of investigation would be to get at the actual mechanisms involved, for example, the FAM136a gene codes for “a mitochondrial protein of unknown function.” Colleagues of ours in Switzerland who study mitochondrial biochemistry asked us for tissue samples from the FAM136a mice and they are now studying these tissues to determine what the function of this protein might be.
Mouse studies with other genes could also shed light on other aspects of the disease, like the tinnitus, the vertigo, or the fluid imbalances leading to endolymphatic hydro’s.
6) How did this collaboration develop?
My collaboration with Dr. Lopez-Escamez developed from a chance encounter we had at the European Brain Prize Meeting in 2012, honoring two famous inner ear female colleagues, one French and the other Israeli, for their outstanding contributions to genetic hearing loss.
I gave a presentation on a hair cell structure called the striated organelle. Dr. Lopez-Escamez approached me afterwards, suggesting a potential identity for a protein component of the striated organelle, that he thought was also involved in MD. It turned out not to be involved in MD, but it was a component of the striated organelle, so he asked us if we wanted to collaborate on localizing some of the gene products he had identified as possible fMD genes in the inner ear. We did localize three of them (FAM136a, DTNA and PRKCB) and published two papers with the Lopez-Escamez laboratory (2015 and 2016).
After the American Hearing Research Foundation came through with funds to purchase the FAM136a and DTNA KO mice and we published last year’s paper with behavioral and biochemical characterizations of those two genes that we had worked on characterizing during the pandemic, we thought that we might be able to get NIH funding for the collaborative work. Most recently, we tried to get NIH funding to create SNP KO mice with more specific mutations, rather than just the global KO mice that we used in our 2023 publication, but so far that has not worked out.
Dr. Lopez-Escamez has since moved from Spain to Australia to become Director of the Center for Meniere’s Disease Research at the University of Sydney. We see each other at international meetings (most recently in Shanghai in April) and continue to consult with each other as much as possible, most recently on the Swiss collaboration. I think that he may have access to more funding and colleagues now in Australia, but we’ll see what the future holds.