No public access
master's thesis
Elucidating the role of optineurin in autophagy

Tereza Ljutić (2016)
Sveučilište u Rijeci
Odjel za biotehnologiju
Metadata
TitleIstraživanje uloge optineurina u procesu autofagije
AuthorTereza Ljutić
Mentor(s)Ivana Munitić (thesis advisor)
Abstract
Optineurin je citoplazmatski protein uključen u procese poput stanične signalizacije, stanične smrti i autofagije. Budući da svoje funkcije obavlja interakcijom s brojnim proteinima, možemo ga smatrati adaptorskim proteinom. Konkretno, u procesu autofagije služi kao prijenosnik sadržaja za razgradnju vežući s jedne strane ubikvitinom obilježeni stanični sadržaj, a s druge strane LC3-II receptor autofagosoma. Također, smatra se da sudjeluje u fuziji autofagosoma sa lizosomom. Poremećena autofagija je jedan od predloženih mehanizama patogeneze amiotrofične lateralne skleroze (ALS), fatalne neurodegenerativne bolesti odumiranja motornih neurona. Mutacije u više od 20 gena su uključene u ALS, od kojih većina, poput mutacija u SOD1, TDP-43 i FUS, ima direktan toksičan učinak na neurone i glija stanice stvarajući unutarstanične proteinske agregate. Suprotno tome, za optineurin se smatra da doprinosi patogenezi ALS-a gubitkom svoje funkcije čime se sugerira njegova neuroprotektivna uloga. U ovom radu smo koristili mišji model optineurina Optn1-470 s deletiranom C-terminalnom ubikvitin-vezujućom domenom (UBD) čime se oponašaju optineurinske UBD mutacije nađene u nekih ALS pacijenata. Cilj nam je bio istražiti učinak optineurinske insuficijencije na autofagiju u primarnim stanicama (makrofazi, mikroglija i neuroni), kao potencijalni uzrok ALS-a. Suprotno očekivanjima, pokazali smo da delecija UBD domene optineurina ne utječe na autofagiju u lipopolisaharidom (LPS)-stimuliranim makrofazima. Sličan rezultat dobili smo i u LPS-om stimuliranoj neonatalnoj mikrogliji, no uz malu tendenciju blokade protoka u Optn1-470 mikrogliji, što još moramo istražiti. Nasuprot tome, preliminarni rezultati u Optn1-470 embrionalnim neuronima su sugerirali smanjen protok bazalne i gladovanjem inducirane autofagije. Također, primijetili smo slabiju motoričku koordiniranost ostarjelih Optn1-470 mužjaka naspram WT kontrola, kao i smanjeni životni vijek. Zaključno, naši rezultati sugeriraju da je Optn1-470 dobar model izučavanja ALS-a te da optineurin potencijalno ima neuroprotektivan učinak putem regulacije autofagije u neuronima. No, zbog drugih predloženih funkcija optineurina, potrebno je provesti daljnja istraživanja kako bi se provjerilo je li potonje uzrok nastanka neurodegeneracije.
Keywordsoptineurin ALS autophagy
Parallel title (English)Elucidating the role of optineurin in autophagy
Committee MembersMiranda Mladinić Pejatović (committee chairperson)
Ivana Munitić (committee member)
Sandra Kraljević Pavelić (committee member)
GranterSveučilište u Rijeci
Lower level organizational unitsOdjel za biotehnologiju
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeMedicinal chemistry
Academic title abbreviationmag. med. chem.
Genremaster's thesis
Language Croatian
Defense date2016-09-30
Parallel abstract (English)
Optineurin is a cytoplasmic protein which was shown to regulate processes like cell signaling, cell death and autophagy. Because it preforms its functions by interacting with many proteins, it is considered to be an adaptor protein. For example, in autophagy, it binds ubiquitinated cargo and bridges it to autophagosomal receptor LC3-II. Also, it is suggested that optineurin plays role in the fusion of autophagosome and lysosome. Dysfunctional autophagy is one of the possible causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons. Mutations in more than 20 genes were described in ALS, with majority of them, like SOD1, TDP-43 and FUS, having direct toxic effect on motor neurons and glial cells through formation of protein aggregates. Conversely, optineurin seems to contribute to ALS pathogenesis by loss-of-function, suggesting it has a neuroprotective role. Here, we used the mouse optineurin model Optn1-470 which lacks the C-terminal ubiquitin-binding domain (UBD), thus mimicking optineurin UBD mutations found in some ALS patients. The goal was to explore the effect of optineurin insufficiency on autophagy in primary cells (macrophages, microglia and neurons) as a possible cause of ALS. Contrary to what we expected, optineurin UBD deletion didn’t affect autophagy in lipopolysaccharide (LPS)-stimulated macrophages. Similar result was obtained in LPS-stimulated neonatal microglia, with only a small tendency for blockade of the autophagy flux in Optn1-470 microglia, which needs further investigation. However, our results in Optn1-470 embryonic neurons suggested a reduced basal autophagy and starvation-induced autophagy. Furthermore, we have shown a reduced motor coordination and survival of aged Optn1-470 male mice. In conclusion, we are suggesting that Optn1-470 is a good ALS model and that optineurin could have a potentially neuroprotective effect via regulating neuronal autophagy. Because optineurin has been linked to many functions, it is imperative to perform additional analyses to determine if the latter is indeed the cause of neurodegeneration.
Parallel keywords (Croatian)optineurin ALS autofagija
Resource typetext
Access conditionNo public access
ProjectProject funder: HRZZ
Project jurisdiction: HR
Project manager: Ivana Munitić
Project name: Istraživanje uloge optineurina u neuroprotekciji
Project acronym: OptineurinNeuroimm
Project ID: 7459
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:919801
CommitterIvana Dorotić