Analiza supstrata enzima BACE1 u endosomalnim frakcijama moždanih regija miševa NPC1-/- i divljeg tipa miša (NCP1+/+)

master's thesis

• Title (english): Distribution of BACE1 substrates in the endosomal fractions isolated from the brain regions of wild type (NCP1+/+) and NPC1-/- disease mouse model
• Author: Sarah Meglaj
• Mentor: Silva Katušić Hećimović (mentor)
• Mentor: Julija Erhardt (sumentor)
• Committee member: Julija Erhardt (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Romana Gračan (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-07-08, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Niemann-Pickova bolest tipa C (NPC) nasljedna je, rijetka neurodegenerativna bolest nakupljanja lipida uzrokovana mutacijama u genu NPC1, a rjeđe NPC2. NPC dijeli niz karakteristika s kompleksnom Alzheimerovom bolesti (AB), ukazujući na postojanje zajedničkih patoloških procesa. Neke od zajedničkih karakteristika su disfunkcija endolizosomalnog puta te nedavno otkrivena povećana proteoliza supstrata β-sekretaze (BACE1) – enzima ključnog u patogenezi AB. Cilj rada bio je ispitati hipotezu da je povećana proteoliza supstrata BACE1 u moždanim regijama miševa NPC1 (NPC1-/-) u odnosu na miševe divljeg tipa (WT, NPC1+/+) posljedica nakupljanja supstrata i/ili enzima BACE1 u endosomima – organelima odgovornima za cijepanje BACE1. Metodom ultracentrifugiranja u diskontinuiranom gradijentu saharoze, izdvojene su frakcije ranih endosoma te kasnih endosoma/lizosoma iz malog mozga i hipokampusa devet tjedana starih miševa NPC1 i WT te su analizirane metodom western blotting, a dobiveni signali kvantificirani su softwareom ImageJ. Rezultati pokazuju različitu distribuciju i razinu biljega ranih endosoma i kasnih endosoma/lizosoma u moždanim regijama NPC1 u odnosu na WT miševe. Također, uočeno je značajno nakupljanje BACE1 supstrata Sez6L u ranim endosomima moždanih regija miševa NPC1 vs. WT. Zaključno, rezultati ukazuju da je Alzheimeru sličan fenotip u bolesti NPC jednim dijelom vjerojatno posljedica poremećaja transporta u endolizosomalnom putu te nakupljanja i povećanog cijepanja supstrata BACE1 u endosomima.

Abstract (english)

Niemann-Pick type C disease (NPC) is a rare, inherited, neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 gene. NPC shares several features with the complex Alzheimer's disease (AD), including endolysosomal dysfunction and the increased proteolysis of the substrates of β-secretase (BACE1), a key protease in AD pathogenesis. The goal of this work was to test the hypothesis that increased proteolysis of BACE1 substrates in brain regions of NPC1 (NPC1-/-) compared to wild-type mice (WT, NPC1+/+) is due to accumulation of BACE1 substrates and/or enzyme BACE1 in endosomes. Ultracentrifugation in a discontinuous sucrose gradient was used to isolate fractions of early and late endosomes/lysosomes from the cerebellum and hippocampus of nine-week-old NPC1 and WT mice. The fractions were analyzed by western blotting, and the obtained signals were quantified using ImageJ software. Different distribution and level of markers of early and late endosomes/lysosomes in the brain regions of NPC1 vs. WT mice was observed. Also, significant accumulation of BACE1 substrate Sez6L was detected in early endosomes in brains of NPC1 mice. In conclusion, the results suggest that the Alzheimer-like phenotype in NPC disease is likely due to endolysosomal dysfunction causing accumulation and increased cleavage of BACE1 substrates in endosomes.

• Keywords: Niemann-Pickova bolest tipa C; Alzheimerova bolest; frakcioniranje; endosomi
• Keywords (english): Alzheimer’s disease; Niemann-Pick type C disease; fractionation; endosomes
• Language: croatian
• URN:NBN: urn:nbn:hr:217:819821
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-07-08
• Created on: 2021-07-22 12:12:06