Periodic Reporting for period 1 - HUNTINFISH (Generation of new zebrafish models for the study of the pathogenesis of Huntington´s disease and for the identification of new therapeutic targets.)
Periodo di rendicontazione: 2018-05-01 al 2020-04-30
The misfolding and aggregation of intracellular proteins is a feature of many late-onset neurodegenerative diseases, called proteinopathies. These include Alzheimer’s disease, Parkinson’s disease, tauopathies and Huntington’s Disease (HD). HD is one of ten known neurodegenerative diseases caused by (CAG)n trinucleotide tract expansions that encode abnormally long polyglutamine (polyQ) tracts and occurs in about 5-10 cases per 100,000 persons. Currently, there are no effective strategies that slow or prevent the neurodegeneration resulting from these diseases in humans. These diseases are predicted to cause increasing economic and social burdens on society particularly as lifespan increases, hence the need for a better understanding of their biology and identification of therapeutic targets. Since HD is a dominant monogenetic disorder, modelling this disease in vitro and in vivo is relatively straightforward and it has been already demonstrated that small molecules that ameliorate HD pathology are also effective in other neurodegenerative diseases, which simply means that the findings in HD models have relevance not only to HD but potentially to all proteinopathies.
Historically, mice have been used to model human diseases because of their physiological, anatomical and genomic similarities to humans. Therefore, the majority of studies in HD have been performed using different mouse models expressing various species of human huntingtin. Although these models have played an important role in providing accurate and experimentally accessible systems to study multiple aspects of disease pathogenesis and to test potential therapeutic treatments, there are aspects that are limiting or even impeding the usage of this model to further study. The zebrafish model has unique characteristics that circumvent many of the limitations found in rodents. As a vertebrate it is excellent for modelling human diseases, it is easy to manipulate pharmacologically and genetically to generate new transgenic lines, its development is fast, its optical transparency facilitates in vivo imaging assays and what is very important it is ideal for high throughput drug screening. Therefore, the main objective of this project is to generate and characterize new zebrafish transgenic lines of HD expressing wild-type and mutant forms of human exon-1 huntingtin, combined with Dendra fluorescent protein, in whole body, neurons and glia and use them to compare the half-lives and in vivo degradation kinetics under basal conditions or under autophagy- or proteasome-inhibition/stimulation conditions. Moreover, we propose to study the role of neuroinflammation and the inflammasomes in the pathogenesis of HD using the newly generated zebrafish transgenic lines, and finally we will test the efficacy of likely protective compounds previously identified in the lab as potential new treatments for HD.
Summarizing the final conclusions of this project, I have been able to successfully generate and characterize zebrafish models for HD, that are clearly showing many aspects of the disease, including huntingtin aggregation, shorter life span, increased neuronal cell death or behavioural hyperactivity. Moreover, I have discovered a novel pathway responsible for accelerated early development in mutant HD fish and I am identifying the mechanism for this which may be suitable to be used in the future as a human disease biomarker. Finally, I have tested in our model five protective compounds, previously identified in the lab, from which two of them has given promising results as a potential new treatment for HD.
Historically, mice have been used to model human diseases because of their physiological, anatomical and genomic similarities to humans. Therefore, the majority of studies in HD have been performed using different mouse models expressing various species of human huntingtin. Although these models have played an important role in providing accurate and experimentally accessible systems to study multiple aspects of disease pathogenesis and to test potential therapeutic treatments, there are aspects that are limiting or even impeding the usage of this model to further study. The zebrafish model has unique characteristics that circumvent many of the limitations found in rodents. As a vertebrate it is excellent for modelling human diseases, it is easy to manipulate pharmacologically and genetically to generate new transgenic lines, its development is fast, its optical transparency facilitates in vivo imaging assays and what is very important it is ideal for high throughput drug screening. Therefore, the main objective of this project is to generate and characterize new zebrafish transgenic lines of HD expressing wild-type and mutant forms of human exon-1 huntingtin, combined with Dendra fluorescent protein, in whole body, neurons and glia and use them to compare the half-lives and in vivo degradation kinetics under basal conditions or under autophagy- or proteasome-inhibition/stimulation conditions. Moreover, we propose to study the role of neuroinflammation and the inflammasomes in the pathogenesis of HD using the newly generated zebrafish transgenic lines, and finally we will test the efficacy of likely protective compounds previously identified in the lab as potential new treatments for HD.
Summarizing the final conclusions of this project, I have been able to successfully generate and characterize zebrafish models for HD, that are clearly showing many aspects of the disease, including huntingtin aggregation, shorter life span, increased neuronal cell death or behavioural hyperactivity. Moreover, I have discovered a novel pathway responsible for accelerated early development in mutant HD fish and I am identifying the mechanism for this which may be suitable to be used in the future as a human disease biomarker. Finally, I have tested in our model five protective compounds, previously identified in the lab, from which two of them has given promising results as a potential new treatment for HD.
Zebrafish models of Huntington’s disease have been generated successfully during the duration of the project. Transgenic fish are expressing human exon-1 of wild-type (23Q) or mutant (74Q) huntingtin together with photoconvertible green-to-red fluorescent protein Dendra in desired tissues (UAS – Gal4 system) allowing in vivo study of protein clearance. The newly established disease model has been carefully studied and analysed and it shares many common features with HD patients. Therefore, this excellent model has a potential to provide insights into human HD with important therapeutic implications.
The development of this project initiated by the characterization of the model, led us to discover for the first time that mutant huntingtin has a role in acceleration of the early development of zebrafish embryos. This finding can potentially be used in the future as an early disease biomarker. Moreover, we have confirmed that mutant transgenic fish have increased neuronal cell death, shorter life span, behavioural hyperactivity and I have shown in vivo mutant huntingtin aggregation.
In order to test if autophagy is an important modifier of pathogenesis in zebrafish as it is in mice, we have tested autophagy up-regulators, which are protective compounds previously identified in the lab to check weather further autophagy increase will be beneficial in the clearance of toxic huntingtin species in vivo. I have shown that two of the compounds gave promising results, confirming that autophagy may be a rational therapeutic strategy for Huntington’s Disease and likewise, this model is appropriate for drug testing with the aim of finding a potential new treatment for HD and other proteinopathies.
All these results have been showed and discussed with the scientific community in different national and international meetings and workshops, and are included in a manuscript that is now in preparation and that will be sent for publication as an open-access article in an international and prestigious scientific journal.
The development of this project initiated by the characterization of the model, led us to discover for the first time that mutant huntingtin has a role in acceleration of the early development of zebrafish embryos. This finding can potentially be used in the future as an early disease biomarker. Moreover, we have confirmed that mutant transgenic fish have increased neuronal cell death, shorter life span, behavioural hyperactivity and I have shown in vivo mutant huntingtin aggregation.
In order to test if autophagy is an important modifier of pathogenesis in zebrafish as it is in mice, we have tested autophagy up-regulators, which are protective compounds previously identified in the lab to check weather further autophagy increase will be beneficial in the clearance of toxic huntingtin species in vivo. I have shown that two of the compounds gave promising results, confirming that autophagy may be a rational therapeutic strategy for Huntington’s Disease and likewise, this model is appropriate for drug testing with the aim of finding a potential new treatment for HD and other proteinopathies.
All these results have been showed and discussed with the scientific community in different national and international meetings and workshops, and are included in a manuscript that is now in preparation and that will be sent for publication as an open-access article in an international and prestigious scientific journal.
Besides the relevant results obtained, the development of this project has also resulted in the creation and optimization of new methods of in vivo imaging, behavioural activity measurements and developmental scoring. Furthermore, multiple zebrafish transgenic lines have been established and characterized, including new reporter lines for HD and several Gal4 driver lines such Ubi:Gal4 giving ubiquitous expression of the reporter or GFAP:Gal4 with glial expression of the reporter. All these tools will be available to the research community upon publication as they may be extremely useful for people working on this field in the future.
Therefore, both the scientific findings and the new techniques resulting from this project will be relevant for researchers in the fields of neuroscience and neurodegeneration. Moreover, these results could have an important socio-economic impact since they can drive to the development of new strategies to treat not only HD but potentially all proteinopathies.
Therefore, both the scientific findings and the new techniques resulting from this project will be relevant for researchers in the fields of neuroscience and neurodegeneration. Moreover, these results could have an important socio-economic impact since they can drive to the development of new strategies to treat not only HD but potentially all proteinopathies.