Yeasts as a cell model for studying Huntinghton's disease

Erzar, Iza (author), Skočaj, Matej (mentor), Xin, Chen (co-mentor)

Huntington's disease (HD) is a neurodegenerative disorder (ND) caused by mutation in huntingtin (HTT) gene. To date there has been no approved cure for HD, which is mainly due to the fact that the molecular mechanisms of the disease have not been well studied and defined. Therefore, stable and reliable model organisms are important for disease studies. Yeast is potential model organism for studying this disease, since both in yeast and in the normal course of the disease, mutated HTT misfolds and forms aggregates within the cell. In this study, yeast cell models stably and constitutively expressing normal and mutated huntingtin (HTT) protein have been constructed. Since yeast has no homolog of huntingtin CRISPR Cas9 system has been used for gene integration. The procedures for phenotypic verification of the models are described, namely growth rate and viability, which includes the determination of CFU (colony-forming-units), spotting assay, and measurement of dead cells using propidium iodide (PI) and flow cytometer. As the GFP (green-fluorescence-protein) is fused at the N-terminal region of HTT protein, its expression and the formation of characteristic aggregates of the mutated protein are observed under the fluorescence microscope. Troubleshooting is also included since difficulties can (and did occur) occur during model construction. The obtained models have great potential as a platform for understanding the disease mechanisms and further enhance their potential for drug screening. Additional research will be needed for the phenotypical characteristics and other properties of these models.

• Language: English
• Keywords: Huntington's diesase, huntingtin, Saccharomyces cerevisiae, yeast, model
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2022
• PID: 20.500.12556/RUL-139038
• COBISS.SI-ID: 119779331
• Publication date in RUL: 29.08.2022

Secondary language

• Language: Slovenian
• Title: Kvasovke kot celični model za preučevanje Huntingtonove bolezni

Abstract

Huntingtonova bolezen (HD) je nevrodegenerativna bolezen (ND), ki je posledica mutacije v genu, ki kodira za protein huntingtin (HTT). Bolezen do danes še nima odobrenega zdravila, kar je predvsem posledica tega, da molekularni mehanizmi poteka bolezni še niso dobro raziskani in definirani. Za preučevanje bolezni so zato pomembni modelni organizmi. Kvasovke so eden izmed potencialnih modelnih organizmov, saj se tako v kvasovki kot v normalnem poteku bolezni mutiran protein HTT napačno zvija in tvori agregate znotraj celice. V raziskavi smo zato pripravili modele kvasovk za preučevanje HD, ki stabilno in konstitutivno izražajo nativen in mutiran protein HTT. Ker kvasovke nimajo homologa huntingtina, je bil za integracijo genov uporabljena metoda CRISPR Cas9. Opisani so postopki fenotipskega preverjanja modelov in sicer hitrost rasti ter viabilnost, kar vključuje določanje CFU (ang., colony-forming-units), kapljični redčitveni test ter barvanje mrtvih celic s propidijevim jodidiom v povezavi s pretočno citometrijo. Protein HTT ima na N-terminalnem koncu dodan zelen fluorescenčni protein (GFP; ang., green-fluorescence-protein), kar nam omogoča opazovanje njegovega izražanja in/ali tvorbo značilnih agregatov mutiranega proteina pod fluorescenčnim mikroskopom. Ker je pridobivanje modelov lahko težavno je to diplomsko delo usmerjeno tudi v procese iskanja in odpravljanja napak v neuspešnih delih priprave celičnih modelov. Pridobljeni modeli imajo velik potencial za nadaljnjo uporabo in sicer pri preučevanju in razumevanju bolezni ter iskanju in preverjanju zdravil. Vsekakor pa bodo potrebne še dodatne raziskave, da se določi fenotipske in druge lastnosti razvitega modela kvasovk.

• Keywords: Huntingtonova bolezen, huntingtin, Saccharomyces cerevisiae, kvasovka, model