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Insights into the Function of Ataxin-3 via CRISPR Gene Knockout.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Insights into the Function of Ataxin-3 via CRISPR Gene Knockout./
作者:	Villegas, Lorenza.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
面頁冊數:	89 p.
附註:	Source: Masters Abstracts International, Volume: 82-10.
Contained By:	Masters Abstracts International82-10.
標題:	CRISPR. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28383973
ISBN:	9798708709516

Insights into the Function of Ataxin-3 via CRISPR Gene Knockout.
Villegas, Lorenza.

Insights into the Function of Ataxin-3 via CRISPR Gene Knockout. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 89 p.

Source: Masters Abstracts International, Volume: 82-10.

Thesis (M.Sc.)--McGill University (Canada), 2020.

This item must not be sold to any third party vendors.

Spinocerebellar Ataxia Type 3 is a rare hereditary neurodegenerative disorder that is the most common form of ataxia in the world. This disease is characterized by a trinucleotide repeat expansion of CAG in the ATXN3 gene, leading to an elongated polyglutamine tract in the ataxin-3 protein. In the disease, the abnormal form of ataxin-3 creates insoluble aggregates that ultimately accumulate in neurons, and whether this accumulation is neuroprotective or toxic, is still being elucidated. In normal conditions, endogenous ataxin-3 has been associated with several cellular pathways including ubiquitination, misfolded protein degradation, DNA damage, transcription, cytoskeletal organization and cell homeostasis, and has been implicated in the interaction with many proteins. Specifically, ataxin-3 interacts and binds with parkin, a protein involved in Parkinson's Disease. Parkin is an E3 ligase in the ubiquitin system and is also involved in mitochondrial quality control, as it removes oxidized cargo and damaged mitochondria from the cell. Ataxin-3 deubiquitinates parkin and impedes its ability to self-ubiquitinate. This project set out to investigate the effect of the absence of ataxin-3 removal on parkin function and stability in the cell as well as in the mitochondria quality control pathway, and the global effect of ataxin-3 absence in the cell. CRISPR-edited HEK293T and U2OSn ataxin-3 KO cell lines revealed successful deletion of ATXN3 gene. Cell cycle analysis seemed normal in these KO lines, showing ataxin-3 removal might not impact cell cycle behavior. However, cell proliferation was reduced in HEK293T ataxin-3 KO cells, demonstrating a possible impact on cell growth. For parkin, immunoblotting and immunofluorescent experiments indicated ataxin-3 KO cells did not have changes in parkin levels, stability, and function. But there was a reduced impact on mitophagy in KO cells in mt-Keima experiment when quantifying mitochondrial turnover. This research revealed that ataxin-3 could have a potential role in mitochondrial quality control with parkin.

ISBN: 9798708709516Subjects--Topical Terms:

3561750
CRISPR.
Subjects--Index Terms:

CRISPR Gene Knockout

Insights into the Function of Ataxin-3 via CRISPR Gene Knockout.
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520 $a Spinocerebellar Ataxia Type 3 is a rare hereditary neurodegenerative disorder that is the most common form of ataxia in the world. This disease is characterized by a trinucleotide repeat expansion of CAG in the ATXN3 gene, leading to an elongated polyglutamine tract in the ataxin-3 protein. In the disease, the abnormal form of ataxin-3 creates insoluble aggregates that ultimately accumulate in neurons, and whether this accumulation is neuroprotective or toxic, is still being elucidated. In normal conditions, endogenous ataxin-3 has been associated with several cellular pathways including ubiquitination, misfolded protein degradation, DNA damage, transcription, cytoskeletal organization and cell homeostasis, and has been implicated in the interaction with many proteins. Specifically, ataxin-3 interacts and binds with parkin, a protein involved in Parkinson's Disease. Parkin is an E3 ligase in the ubiquitin system and is also involved in mitochondrial quality control, as it removes oxidized cargo and damaged mitochondria from the cell. Ataxin-3 deubiquitinates parkin and impedes its ability to self-ubiquitinate. This project set out to investigate the effect of the absence of ataxin-3 removal on parkin function and stability in the cell as well as in the mitochondria quality control pathway, and the global effect of ataxin-3 absence in the cell. CRISPR-edited HEK293T and U2OSn ataxin-3 KO cell lines revealed successful deletion of ATXN3 gene. Cell cycle analysis seemed normal in these KO lines, showing ataxin-3 removal might not impact cell cycle behavior. However, cell proliferation was reduced in HEK293T ataxin-3 KO cells, demonstrating a possible impact on cell growth. For parkin, immunoblotting and immunofluorescent experiments indicated ataxin-3 KO cells did not have changes in parkin levels, stability, and function. But there was a reduced impact on mitophagy in KO cells in mt-Keima experiment when quantifying mitochondrial turnover. This research revealed that ataxin-3 could have a potential role in mitochondrial quality control with parkin.
520 $a L'ataxie spinocerebelleuse de type 3 est la forme la plus frequente d'ataxie au monde. C'est une maladie neurodegenerative hereditaire rare, caracterisee par une expansion anormale du triplet de nucleotides CAG dans le gene ATXN3. Cette repetition se traduit par la presence d'une expansion polyglutaminique dans la proteine ataxine-3, entrainant la formation d'agregats insolubles de la proteine anormale, qui s'accumulent dans les neurones. La nature neuroprotectrice ou toxique de ces agregats reste a ce jour non determinee. Dans sa forme normale, la proteine ataxine-3 interagit avec de nombreuses autres proteines, et est associee a differents mecanismes cellulaires telles que la voie d'ubiquitination, la degradation des proteines mal repliees, l'alteration de l'ADN, la transcription, l'organisation du cytosquelette, ou encore le maintien de l'homeostasie cellulaire. L'ataxine-3 interagit avec la proteine parkine, qui joue un role dans la maladie de Parkinson. La parkine est une enzyme de type ubiquitine ligase E3 impliquee dans le controle qualite des mitochondries, permettant la degradation des proteines cargo oxydees et des mitochondries dysfonctionnelles.L'ataxine-3 est responsable de la deubiquitination de la parkine et empeche son auto-ubiquitination. L'objectif de ce travail est d'etudier l'impact de l'absence de l'ataxine-3 sur la stabilite et la fonction de la parkine et sur le controle qualite des mitochondries, et plus generalement l'effet de son absence au niveau de la cellule. Lors de ce travail, des lignees de cellules HEK-293T et U2OSn ataxin-3 KO, depletees de l'expression de l'ataxine-3 par la technique d'edition de genome CRISPR, ont ete generees. Le cycle cellulaire de ces cellules est normal mais la proliferation des cellules HEK-293T ataxin-3 KO est diminuee, demontrant que la proteine ataxin-3 ne serait pas impliquee dans la regulation du cycle cellulaire, mais jouerait un role potentiel dans la proliferation des cellules. Des analyses par immunoblot et immunocytochimie dans ces memes cellules ont mis en evidence que l'absence d'expression de l'ataxine-3 n'a pas d'effet sur le niveau d'expression, sur la stabilite, ou sur la fonction de la parkine. Enfin, l'analyse de la mitophagie dans des cellules ataxin-3 KO exprimant la proteine mito-Keima a montre une faible diminution de celle-ci. Cette recherche a ainsi mis en evidence le role potentiel de l'ataxine-3 dans le controle qualite des mitochondries, en association avec la parkine.
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