The project aims to experimentally develop an integrated and automated solution for screening drugs and genetic interventions for neurodegenerative diseases, using the nematode C. elegans and ageing-related data.
Ageing is the major risk factor for neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. The graying of the population has been accompanied by a continual increase in the prevalence of neurodegeneration amongst the elderly and is now one of the greatest challenges that healthcare and research has to tackle. In this project, we propose to experimentally develop an integrated and automated solution for screening drugs and genetic interventions for neurodegenerative diseases, using the nematode C. elegans and ageing-related data. The system will include: 1) a network-based, integrative, and predictive bioinformatics method for short-listing genetic targets of interest, and 2) a low-cost, modular, automated video-recording platform for healthspan screening in worms. Additionally, we also propose the implementation of a gene activation system that would complement the existing genetic tools for investigating gene function and drug screening in worms, by creating a CRISPR-based overexpression system with the flexibility of RNA interference approaches. Lastly, we propose to validate the developed system by monitoring predicted interventions, both using the automated platform and by traditional lab methods.
The overall goal of the project is to build an automated system for phenotypic screening in nematodes which will be used to test predicted targets for neurodegenerative diseases. More specifically, the objectives are: 1) Predicting using a systems biology approach molecular targets relevant to ageing and neurodegeneration; 2) Building a low-cost, modular, video-monitoring system for nematodes, capable of simultaneously assaying lifespan and healthspan features for a large number of plates with worm cultures; 3) Expanding the current capacity of molecular interventions in worms with a method that allows the overexpression of specific targets, controlled through food-delivered dCAS9 CRISPR guides; and 4) Testing and validating the developed system by monitoring predicted interventions and comparing with traditional lab methods (manually culturing worms and observing their phenotype).
Upon the implementation of the project, the following outcomes should be achieved: 1) the group will have a functional prototype system (at the experimental proof of concept level) that allows with minimal human interventions to survey the lifespan and healthspan of multiple worm culture plates; 2) the group will have a validated protocol to overexpress genes in C. elegans using CRISPR; 3) the group will have evaluated phenotype changes for several interventions in relation to neurodegeneration.
Vârsta este factorul major de risc în cazul patologiilor precum boala Alzheimer sau boala Parkinson. Creșterea semnificativă a segmentului de populație vârstnică a fost prin urmare acompaniată și de o continua creștere a prevalenței patologiilor neurodegenerative în randul populatiei și este acum una dintre marile provocări ale sistemului de sănătate sau de cercetare.
În acest proiect am dezvoltat unelte pentru predicția și evaluarea cât mai automatizată a medicamentelor și intervențiilor genetice în cazul bolilor neurodegenerative, folosind nematodul C. elegans și date legate de îmbătrânire. Au fost realizate analize de bioinformatică, pornind de la date de genetică și farmacologie, obtinandu-se o prioritizare a unor medicamente cu potențial în boala Alzheimer. Aceste medicamente au fost testate individual sau în combinatie într-un model de nematode (viermisori C. elegans) ce mimează mecanisme degenerative cauzate de acumularea beta-amiloidului - una din principalele caracteristici în Alzheimer.
Pentru o testare mai eficienta a fost dezvoltată și o platformă automatizată de monitorizare video, cu costuri reduse și modulară, pentru screeningul stării de sănătăte la C. elegans, platforma ce speram sa fie folosită într-o mult mai largă gama de aplicații. În plus, aceasta platforma va fi complementată de un sistem de supra-activare a genelor (dezvoltat tot în acest proiect și bazat pe tehnologia CRISPR-activation), care să complementeze instrumentele genetice existente (cum ar fi silentierea prin ARN). Folosind aceste doua unelte (monitorizare automatizata si intervenții moleculare - fie de supraexprimare, fie de silențiere), se pot realiza experimente mult mai ample, cu un număr mult mai mare de cazuri (ex: tratament cu medicamente, intervenții genetice, etc) evaluate simultan.
Platforma dezvoltata pentru monitorizarea populatiilor de nematode
Rezultate (încă nepublicate) despre îmbunătățirea fenotipului motoriu al nematodelor model pentru Alzheimer, prin folosirea sinergica a doua medicamente. De la stanga la dreapta, control, efectul individual al celor 3 medicamente, efectul combinatiilor de cate 2 medicamente, efectul cocktail-ului de 3 medicamente.
Example of a video capture with marking of the software tracking of a well with nematodes (WT) in a 24 well plate.
Summary of results obtained in 2020:
In 2020, we have prepared the data for the project and started the bioinformatics analysis to predict genes that will be genetically manipulated in C. elegans for neurodegenerative disorders. This has resulted in a preliminary list of genes that are involved in each of the studied pathologies and in a gene prioritization based on their importance in gene networks associated with these disorders. This activity will continue in the next phase (2021) as well.
Additionally, we have started work on the video monitoring prototype for nematodes, putting most of the effort into the development of the initial static rig. This has been followed up by several rounds of initial testing. For the multi-plates system, a technical design has been already carried out and an implementation plan formulated.
Lastly, we have started working on the overexpression method based on dCAS9 CRISPR for nematodes.
So far, all the activities in the project are according to the proposal's GANTT.
Summary of results obtained in 2021:
In 2021, several bioinformatics analyses have been carried out resulting in a set of molecular targets relevant for Alzheimer's and Parkinson's diseases. Part of these targets will be experimentally validated starting 2022.
Additionally, progress has been achieved in the automatization of lifespan and healthspan monitoring, with the static rig being fully functional and work at the 1 layer (and even multi-layer) monitoring system being underway.
A method of genetic overexpression in C. elegans has also been developed.
We would also like to mention that part of the bioinformatics results have been organized in a scientific paper, which is currently in press. In this article, a systematic analysis of small molecules involved in induced pluripotency has been done and the links of such molecules with aging and age-related pathological conditions (including neurodegenerative disorders) have been investigated.
Currently, all the activities in the project are on track and according to the proposal's initial GANTT.
Summary of results obtained in 2022:
In 2022, the bioinformatics predictions were validated and the development of a modular prototype for the phenotypic monitoring and screening system was completed. For validation, a screening was performed in the case of pharmaceutical or genetically modified model animals and the analysis of the involvement of these targets in neurodegenerative diseases was carried out.
In more detail, in this phase of the project: