Title Interactions between schizophrenia-associated aggregation of EHD3 and the cytoskeleton Title (croatian) Interakcije između agregacije EHD3 proteina povezane sa schizofrenijom i citoskeleta Author Andrea Montan Mentor Nicholas Bradshaw (mentor)Committee member Nicholas Bradshaw (predsjednik povjerenstva)Committee member Anđelka Radojčić Badovinac (član povjerenstva)Committee member Antonija Jurak Begonja (član povjerenstva)Granter University of Rijeka Defense date and country 2024-09-24, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Schizophrenia is a serious mental illness that is characterized by altered
behaviour and a diminished ability to perceive reality. In addition to genetic
and environmental risk factors, protein aggregation is also considered to be
a potential risk factor in the development of schizophrenia. Protein
aggregation and misfolding happens when cellular systems are disturbed by
internal and external stresses. One of the proteins that has been found to
aggregate in the brains of schizophrenia patients is EH-domain containing
3 (EHD3). This belongs to the C-terminal Eps15 homology domain (EHD)
protein family, together with the EHD1, EHD2 and EHD4 proteins. EHD3 is
involved in the regulation of endosome to Golgi transport, recycling, and in
promotion of dopaminergic transmission. Previous research in our lab
showed that full length EHD3 aggregated in SH-SY5Y cells, while two
truncated versions, containing amino acids 1-399 and 1-434 did not. Also,
the EH domain alone (amino acids 435-535), when expressed individually
did not aggregate. The conclusion was that more than one region influences
EHD3 aggregation. The EH domain is necessary for the initialization of EHD3
aggregation, but other structural regions interact and recruit other
molecules that make up the aggregates. In my thesis, we wanted to test
the association of EHD3 with actin and how it affects the cytoskeleton. We
also wanted to determine how other proteins from EHD family behave inside
cells and whether they also form aggregates. Using ultracentrifuge assay, I
saw that full length EHD3 co-segregates and is connected with actin inside
cells, which I also confirmed using fluorescent microscopy. In
ultracentrifuge assay, EHD3 fragments 1-399 and 1-434 did not show
association with actin, while in fluorescent microscopy they showed
association with actin cytoskeleton. Further research is needed in this field.
In blinded, quantified, microscopy assay, EHD3 showed aggregate-like
structures in most of the cells, while EHD1 showed this pattern in
approximately half of cells. EHD2 and EHD4 showed low signs of potential
aggregation. From this we can conclude that, while EHD3 is the only EHD
protein confirmed to aggregate, data for EHD1 show its potential for
aggregation. Understanding of aggregation of EHD proteins processes and
physiological functions may facilitate a better understanding of the
pathology and possible causes of schizophrenia development
Abstract (croatian) Shizofrenija je ozbiljna mentalna bolest koju karakterizira promjena
ponašanja i smanjena sposobnost percepcije stvarnosti. Osim genetskih i
okolišnih rizičnih čimbenika, agregacija proteina također se smatra
potencijalnim čimbenikom za razvoj shizofrenije. Agregacija i pogrešno
slaganje proteina događa se kada su stanični sustavi i procesi poremećeni
unutarnjim i vanjskim stresovima. EHD3 je jedan od proteina za koji je
utvrđeno da agregira u mozgu pacijenata sa shizofrenijom. Taj protein
pripada obitelji proteina C-terminalne homološke domene Eps15 (EHD),
zajedno s proteinima EHD1, EHD2 i EHD4. EHD3 je uključen u regulaciju
transporta endosoma u Golgijevo tijelo, recikliranje te promicanje prijenosa
dopaminergika. Prethodna istraživanja u našem laboratoriju dokazala su da
EHD3 agregira u SH-SY5Y stanicama, dok njegove dvije skraćene verzije (s
aminokiselinama 1-399 i 1-434) ne agregiraju. Također, kada se EH domena
(aminokiseline 435-535) ekspresirala pojedinačno u stanici, nije pokazala
znakove agregacije. Donesen je zaključak da je više regija uključeno u
proces agregacije EHD3 proteina. EH domena neophodna je za inicijalizaciju
agregacije, ali druge strukturne regije međusobno djeluje i regrutiraju
druge molekule koje čine agregate. U ovom smo radu željeli testirati
povezanost EHD3 proteina s aktinom te utvrditi kako utječe na citoskelet
stanica. Također, promatrali smo kako se drugi proteini iz EHD obitelji
ponašaju unutar stanica i tvore li agregate. Koristeći ultracentrifugu, otkrila
sam da EHD3 protein ko-agregira te je povezan s aktinom unutar stanica.
Isto sam utvrdila i uz pomoć fluorescentne mikroskopije. Kod testa
ultracentrifuge, EHD3 fragmenti 1-399 i 1-434 nisu pokazali povezanost a
aktinom dok su kod fluorescentne mikroskopije pokazali povezanost s
aktinskim citoskeletom. U ovom su području potrebna daljnja istraživanja.
U slijepom, kvantificiranom testu mikroskopije, EHD3 protein pokazao je
strukture slične agregatima u većini stanica dok je EHD1 pokazao taj
obrazac kod polovice stanica. S druge strane, EHD2 i EHD4 proteini pokazali
su male znakove potencijalne agregacije. Iz ovih rezultata možemo
zaključiti da, iako je EHD3 jedini EHD protein za koji je potvrđeno da
agregira, podaci za EHD1 pokazuju njegov potencijal za agregaciju.
Razumijevanje procesa agregacije i fizioloških funkcija EHD proteina može
olakšati bolje razumijevanje patologije te omogućiti bolje razumijevanje
uzroka razvoja shizofrenije.
Keywords
schizophrenia
EHD3
cytoskeleton
Keywords (croatian)
schizofrenija
EHD3
citoskelet
Language english URN:NBN urn:nbn:hr:193:436901 Study programme Title: Drug research and development Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2024-11-04 11:47:50
