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The effect of mutant SOD1 on cell viability

(2016) Boertien, L.M.

Mutations in the gene encoding for superoxide dismutase 1 (mSOD1) lead to protein aggregation and
are associated with 20% of familial amyotrophic lateral sclerosis (ALS) cases. Multiple molecular
chaperones are known to influence mSOD1 aggregation and cytotoxicity. Still, the relation between
protein aggregation and cytotoxicity remains elusive.
In the current project, human osteosarcoma cell lines stably expressing SOD1-WT or SOD1-A4V
(U2OS-WT, U2OS-A4V) were exposed to endoplasmatic reticular stress, proteasomal inhibition and
autophagy inhibition. Phenotypes of SOD1-A4V cytotoxicity were then correlated to levels of mSOD1
expression and aggregation. Additionally, overexpression of the molecular chaperones HSPA1A and
HSPA1L was investigated for their potential to rescue the mSOD1 associated phenotype.
Endoplasmatic reticular (ER) stress induced by Tunicamycin treatment was the only stressor that
produced a reproducible phenotype characterised by a decrease in U2OS-A4V cell viability compared
to U2OS-WT. No correlation with mSOD1 aggregation could be established. HSPA1A and HSPA1L
overexpression both resulted in a marginal relative rescue of cell viability specific to U2OS-A4V under
Tunicamycin treatment.
Interpretation of the results was hampered by the marginal differences in cell viability, the low
expression levels of SOD1-A4V and the expression of endogenous SOD1. An experimental
endogenous SOD1 knockout model, with expression levels of the proteins of interest close to
endogenous expression of SOD1, is proposed to substantiate the findings of this study. Further
investigation of the relationship between ER stress and mSOD1 cytotoxicity can be centred around
the in literature described interaction between mSOD1 and Derlin-1, through which mSOD1 is
proposed to mediate its cytotoxicity.





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ID 3344
Moeder ID 3084
Volgorde Boertien, L.M.
Naam BoertienLM
Publiceren yes
OAI-naam Student_thesis
Path root/geneeskunde/2016/BoertienLM/
Gemaakt op: 2017-02-21 12:44:55
Gemodificeerd op: 2017-02-21 12:44:55
Digitaal ID 58ac36463abb1
Afstudeerrichting opleiding/afstudeerrichting 1
Studierichting Studierichting 1
Titel The effect of mutant SOD1 on cell viability
Ruilverkeer mogelijk no
Printen in opdracht no
Aantal pagina's 25
Publicatiejaar 2016
Taal en
Engelse samenvatting Mutations in the gene encoding for superoxide dismutase 1 (mSOD1) lead to protein aggregation and
are associated with 20% of familial amyotrophic lateral sclerosis (ALS) cases. Multiple molecular
chaperones are known to influence mSOD1 aggregation and cytotoxicity. Still, the relation between
protein aggregation and cytotoxicity remains elusive.
In the current project, human osteosarcoma cell lines stably expressing SOD1-WT or SOD1-A4V
(U2OS-WT, U2OS-A4V) were exposed to endoplasmatic reticular stress, proteasomal inhibition and
autophagy inhibition. Phenotypes of SOD1-A4V cytotoxicity were then correlated to levels of mSOD1
expression and aggregation. Additionally, overexpression of the molecular chaperones HSPA1A and
HSPA1L was investigated for their potential to rescue the mSOD1 associated phenotype.
Endoplasmatic reticular (ER) stress induced by Tunicamycin treatment was the only stressor that
produced a reproducible phenotype characterised by a decrease in U2OS-A4V cell viability compared
to U2OS-WT. No correlation with mSOD1 aggregation could be established. HSPA1A and HSPA1L
overexpression both resulted in a marginal relative rescue of cell viability specific to U2OS-A4V under
Tunicamycin treatment.
Interpretation of the results was hampered by the marginal differences in cell viability, the low
expression levels of SOD1-A4V and the expression of endogenous SOD1. An experimental
endogenous SOD1 knockout model, with expression levels of the proteins of interest close to
endogenous expression of SOD1, is proposed to substantiate the findings of this study. Further
investigation of the relationship between ER stress and mSOD1 cytotoxicity can be centred around
the in literature described interaction between mSOD1 and Derlin-1, through which mSOD1 is
proposed to mediate its cytotoxicity.
Nederlandse samenvatting geassocieerd met 20% van de familiaire gevallen van amyotrofische laterale sclerose (ALS). Meerdere
moleculaire chaperones hebben invloed op mSOD1 aggregatie en toxiciteit. Desondanks is de relatie
tussen mSOD1 aggregatie en cytotoxiciteit nog grotendeels onbekend.
In het huidige project werden humane osteosarcoom cellijnen met een stabiele expressie van SOD1-
WT en SOD1-A4V (U2OS-WT, U2OS-A4V) blootgesteld aan verschillende stressoren (endoplasmatisch
reticulaire stressoren, proteasoom inhibitie, autofagie inhibitie) om een fenotype van SOD1-A4V
toxiciteit te bewerkstelligen. Deze werd indien mogelijk gecorreleerd aan mSOD1 expressie en
aggregatie. Vervolgens werd middels overexpressie onderzocht in hoeverre de moleculaire
chaperones HSPA1A en HSPA1L de potentie hebben om het gecreëerde fenotype te redden.
Alleen Tunicamycine, een endoplasmatisch reticulaire (ER) stressor, produceerde een
reproduceerbaar fenotype welke werd gekenmerkt door relatief verminderde celviabiliteit van U2OSA4V
ten opzichte van U2OS-WT. Dit fenotype kon niet worden gecorreleerd aan mSOD1 aggregatie.
Overexpressie van HSPA1A en HSPA1L resulteerde in beide gevallen tot een relatieve toename van
de celviabiliteit, specifiek voor U2OS-A4V onder de behandeling met Tunicamycine.
De interpretatie van deze resultaten wordt gehinderd door de kleine verschillen in celviabiliteit, de
lage U2OS-A4V expressie en de aanwezigheid van endogeen SOD1. Een experimenteel model zonder
endogeen SOD1 en expressie niveaus van SOD1-WT en -A4V vergelijkbaar met endogene expressie
wordt voorgesteld om de resultaten van deze studie te onderbouwen. Verder onderzoek naar de
relatie tussen mSOD1 toxiciteit en ER-stress dient gericht te zijn op de in de literatuur beschreven
interactie tussen mSOD1 en Derlin-1, welke de toxiciteit van mSOD1 mogelijk medieert.
Onderwijsinstelling Medical Sciences
Type embargo abstract openbaar, scriptie op aanvraag
Auteur(s) Boertien, L.M.
UMCG begeleider(s) Faculty Supervisor:; Bergink, S. (Steven); Secondary Supervisor:; Serlidaki, D. (Despina); Department of Medical Cell Biology UMCG
Auteur(s) Boertien, L.M.
UMCG begeleider(s) Faculty Supervisor:; Bergink, S. (Steven); Secondary Supervisor:; Serlidaki, D. (Despina); Department of Medical Cell Biology UMCG


 
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