Periodic Reporting for period 1 - FLYMEN (A Drosophila model for understanding the basis of Meniere’s disease)
Reporting period: 2019-01-07 to 2021-01-06
Around 12 in 1000 people in the world will have balance issues and hearing loss due to a condition called Meniere´s disease (MD). The cause of Meniere´s disease is unknown. It typically begins with vertigo attacks in young adults and then leads to hearing loss in thirties or forties. Symptoms vary between people and over time. The main problems are unpredictable attacks of vertigo that can last from a few minutes to 24 hours. Consequently, Meniere´s disease is an unpredictable and distressing illness. Currently the only treatment is reducing and controlling symptoms (anti-vertigo medications and hearing aids). Moreover, there is no specific test that, on its own, is reliable in diagnosing Meniere’s disease and the symptoms overlap with many other illnesses. So the final diagnosis normally takes several months or even years.
The problems of treatment and diagnosis are due to the fact that Meniere's disease is complex and the underlying causes are unknown. Many factors are thought to be involved in the development of the condition, such as increased pressure of fluid in the inner ear. There is an inherited component - and some candidate defective genes have been identified in families with the disease. It is thought that environmental triggers are also important. The relationship between these factors and the progression of the disease however remains unclear. Unfortunately, despite many efforts to understand the genetic and molecular basis of the disease, results remain limited due to the absence of a suitable in vivo model.
The worldwide incidence suggests that around 100,000 patients develop MD every year. But MD is complex and difficult to diagnose. Currently treatment is confined to treating symptoms. Lack of understanding of basic aetiology impairs rational progress in diagnosis and treatment.
MD is considered a debilitating disease that interferes with the activities of daily living and imposes substantial costs to national health systems due to the lack of effective treatment. Overall, the use of animal models of MD will enable understand of cells, pathways and trigger involve in MD and could open several lines of translational research including a novel research field focus on discovering of new target and more accurate drugs to beneficial the patient health.
The goal of FLYMEN is to create a new animal model for MD, with the fruit fly (Drosophila), and thereby identify pathways involved in the MD-associated decline of auditory and vestibular function and to probe the interactions between genetic and environmental factors. To this end, I will pursue three major aims: 1) Does the deletion of key genes in Drosophila produce hearing loss and/or vestibular phenotypes with similarities with human MD? 2) What is the basis of MD-related degeneration? This will be assessed by expression and morphological analyses of mutants. 3) Do environmental factors trigger or enhance MD-related degeneration?
Conclusions of the action
The FLYMEN project has investigated the role of several MD candidate genes inside the Drosophila ear, biased towards pathway or molecular discovery and consequently a treatment. The exploration of these genes in Drosophila resulted in first animal model with MD-like phenotype without any drug conditioning knocking out the Dyb gene. In addition we are generated a Dyb-GFP enhancer transgenic reporter line that has allow the identification of cells involved.
The problems of treatment and diagnosis are due to the fact that Meniere's disease is complex and the underlying causes are unknown. Many factors are thought to be involved in the development of the condition, such as increased pressure of fluid in the inner ear. There is an inherited component - and some candidate defective genes have been identified in families with the disease. It is thought that environmental triggers are also important. The relationship between these factors and the progression of the disease however remains unclear. Unfortunately, despite many efforts to understand the genetic and molecular basis of the disease, results remain limited due to the absence of a suitable in vivo model.
The worldwide incidence suggests that around 100,000 patients develop MD every year. But MD is complex and difficult to diagnose. Currently treatment is confined to treating symptoms. Lack of understanding of basic aetiology impairs rational progress in diagnosis and treatment.
MD is considered a debilitating disease that interferes with the activities of daily living and imposes substantial costs to national health systems due to the lack of effective treatment. Overall, the use of animal models of MD will enable understand of cells, pathways and trigger involve in MD and could open several lines of translational research including a novel research field focus on discovering of new target and more accurate drugs to beneficial the patient health.
The goal of FLYMEN is to create a new animal model for MD, with the fruit fly (Drosophila), and thereby identify pathways involved in the MD-associated decline of auditory and vestibular function and to probe the interactions between genetic and environmental factors. To this end, I will pursue three major aims: 1) Does the deletion of key genes in Drosophila produce hearing loss and/or vestibular phenotypes with similarities with human MD? 2) What is the basis of MD-related degeneration? This will be assessed by expression and morphological analyses of mutants. 3) Do environmental factors trigger or enhance MD-related degeneration?
Conclusions of the action
The FLYMEN project has investigated the role of several MD candidate genes inside the Drosophila ear, biased towards pathway or molecular discovery and consequently a treatment. The exploration of these genes in Drosophila resulted in first animal model with MD-like phenotype without any drug conditioning knocking out the Dyb gene. In addition we are generated a Dyb-GFP enhancer transgenic reporter line that has allow the identification of cells involved.
AIM 1. Does the deletion of key genes in Drosophila produce hearing loss and/or vestibular phenotypes with similarities with human MD?
The first objective focussed on identifies hearing loss and/or vestibular phenotype of MD genes using knockout. Since some of the genes are broadly expressed in and may be involved in different functions, the phenotype of full knockout flies could be due to a non-auditory neuron function. Therefore, to differentiate between proprioceptive neuron and other defects, we used Gal4-driven RNAi lines to generate tissue- and cell-type-specific knockdown. These additionally facilitated identifying the auditory JO cell-types responsible for phenotypes observed. The auditory/vestibular phenotypes of mutant flies were determined through functional test including analyses of unstimulated sound receivers by Laser Doppler Vibrometry (LDV) (which tests auditory response) and climbing assay (which test proprioceptive ability). Both methods were previously well stabled by Prof. Jarman, who has an international reputation in deciphering the transcriptional logic of sensory maintenance in Drosophila, and Dr Albert, an internationally renowned expert in sensory biology and biophysics.
Main results achieved:
1. Established that MD candidate gene DTNA/dyb indeed shows an MD-like phenotype in flies.
2. Gal4-driven RNAi lines suggest that neurons could be affected.
3. LDV evidence that both sensitive and insensitive channels are altered.
AIM 2. What is the basis of MD-related degeneration?
The second objective was based on identifying responsible cell types and unravelling cellular defects, molecular mechanisms and pathways. To do this, special focus was given to the development of 1) immunofluorescence on mutants to determine defects and 2) Dyb-GFP enhancer transgenic reporter line, to screen Dyb location in the Drosophila antenna. In addition the fellow obtained confidence in the use of well-established expression pattern techniques: (i) RNA in situ hybridisation; (ii) Generation of transgenic reporter line (injecting expression vector in embryos), dissection of endorgans and immunolabeling to perform confocal microscopy from flies.
Main results achieved:
1. Established Dyb-GFP enhancer transgenic reporter line.
2. The transgenic reporter line show strong co-localization of GFP with a ligament cell marker (Imagen 1).
3. One of the ion channel (NompC) showed a aberrantly distribution at the tip of the JO neuron dendrites.
4. A transgenic line carrying Iav-GFP fusion protein and Dyb knockout (w: Dyb11, Iav-GFP) was established.
Dissemination and public engagement
The researcher attended to the 26th European Drosophila Research Conference (EDRC) on 2019, 62th Annual Drosophila Research Conference (ADRC), both with poster presentation. Also she present in the ARO 43st International midwinter meeting with an oral communication. Additionally, during the course of this fellowship she has planned to attend the Barany meeting as Invited speaker and as part of the organising committee of one of the Symposiums inside the meeting. However this has been postponed to 2022.
The first objective focussed on identifies hearing loss and/or vestibular phenotype of MD genes using knockout. Since some of the genes are broadly expressed in and may be involved in different functions, the phenotype of full knockout flies could be due to a non-auditory neuron function. Therefore, to differentiate between proprioceptive neuron and other defects, we used Gal4-driven RNAi lines to generate tissue- and cell-type-specific knockdown. These additionally facilitated identifying the auditory JO cell-types responsible for phenotypes observed. The auditory/vestibular phenotypes of mutant flies were determined through functional test including analyses of unstimulated sound receivers by Laser Doppler Vibrometry (LDV) (which tests auditory response) and climbing assay (which test proprioceptive ability). Both methods were previously well stabled by Prof. Jarman, who has an international reputation in deciphering the transcriptional logic of sensory maintenance in Drosophila, and Dr Albert, an internationally renowned expert in sensory biology and biophysics.
Main results achieved:
1. Established that MD candidate gene DTNA/dyb indeed shows an MD-like phenotype in flies.
2. Gal4-driven RNAi lines suggest that neurons could be affected.
3. LDV evidence that both sensitive and insensitive channels are altered.
AIM 2. What is the basis of MD-related degeneration?
The second objective was based on identifying responsible cell types and unravelling cellular defects, molecular mechanisms and pathways. To do this, special focus was given to the development of 1) immunofluorescence on mutants to determine defects and 2) Dyb-GFP enhancer transgenic reporter line, to screen Dyb location in the Drosophila antenna. In addition the fellow obtained confidence in the use of well-established expression pattern techniques: (i) RNA in situ hybridisation; (ii) Generation of transgenic reporter line (injecting expression vector in embryos), dissection of endorgans and immunolabeling to perform confocal microscopy from flies.
Main results achieved:
1. Established Dyb-GFP enhancer transgenic reporter line.
2. The transgenic reporter line show strong co-localization of GFP with a ligament cell marker (Imagen 1).
3. One of the ion channel (NompC) showed a aberrantly distribution at the tip of the JO neuron dendrites.
4. A transgenic line carrying Iav-GFP fusion protein and Dyb knockout (w: Dyb11, Iav-GFP) was established.
Dissemination and public engagement
The researcher attended to the 26th European Drosophila Research Conference (EDRC) on 2019, 62th Annual Drosophila Research Conference (ADRC), both with poster presentation. Also she present in the ARO 43st International midwinter meeting with an oral communication. Additionally, during the course of this fellowship she has planned to attend the Barany meeting as Invited speaker and as part of the organising committee of one of the Symposiums inside the meeting. However this has been postponed to 2022.
The fellow learnt state-of-the-art techniques in Drosophila genetics, behavioural assays and biophysics associated with hearing and balance becoming confident in using a diverse range of tools and experiments. She, indeed, acquired the knowledge to independently carry out the two main objectives from this Marie Curie action. The results of the project have been presented in international conferences as both oral and poster presentation and it is under preparation for submission while the third objective was postponed due SARS-CoV-2 outbreak.
Despite the preclinical stage of the results they are increased the understanding of how and why genes influence Meniere disease development. In addition, this study has developed in first animal model with MD-like phenotype without any drug conditioning. These findings will provide a springboard for rational clinical research into diagnosis and treatments.
Despite the preclinical stage of the results they are increased the understanding of how and why genes influence Meniere disease development. In addition, this study has developed in first animal model with MD-like phenotype without any drug conditioning. These findings will provide a springboard for rational clinical research into diagnosis and treatments.