Title Karakterizacija neuropatoloških značajki novog mišjeg modela bolesti Niemann-Pick tipa C s deletiranim genom BACE1 Title (english) Characterisation of neuropathological features of a new Niemann-Pick type C disease murin model with genetically depleted BACE1 Author Lea Vidatić Mentor Silva Katušić Hećimović (mentor)Committee member Jelena Ban (predsjednik povjerenstva)Committee member Željka Minić (član povjerenstva)Committee member Dubravka Švob Štrac (član povjerenstva)Granter University of Rijeka Defense date and country 2024-05-24, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Bolest Niemann-Pick tipa C (NPC) je rijetka monogenska neurodegenerativna bolest i
poremećaj nakupljanja lizosoma. Bolest NPC uzrokovana je mutacijom u genima NPC1 ili
NPC2 koji kodiraju za prijenosnike kolesterola. Zbog nedostatka ili promijenjene funkcije
proteina NPC1 ili NPC2, u kasnim endosomima i lizosomima dolazi do nakupljanja
slobodnog kolesterola, što uzrokuje poremećaj funkcije endolizosomalnog puta, snažnu
neuroinflamaciju (aktivaciju astrocita i mikroglija) i neurodegeneraciju prvenstveno
Purkinjeovih neurona u malom mozgu. Klinički, bolest NPC karakterizira heterogeni fenotip
koji uključuje brojne neurovisceralne poremećaje i ranu smrt. Unatoč etiološkoj razlici
između bolesti NPC i kompleksne Alzheimerove bolesti (AB), ovi poremećaji dijele nekoliko
patoloških značajki, poput promijenjenog metabolizma kolesterola, neurodegeneracije,
neuroinflamacije te nakupljanja hiperfosforiliranog proteina tau i peptida amiloida-β u
mozgu. Povećano stvaranje amiloida-β uslijed povećanog proteolitičkog cijepanja proteina
APP enzimom BACE1 smatra se glavnim mehanizmom patogeneze AB. Zanimljivo,
povećana proteolitička aktivnost enzima BACE1 zabilježena je i u bolesti NPC. S obzirom
da je enzim BACE1 potencijalna farmakološka meta za liječenje AB, cilj ovog doktorskog
rada bio je ispitati učinak genetičke modulacije razine enzima BACE1 na razinu i progresiju
neuropatoloških značajki bolesti NPC. Za ovo istraživanje korišten je mišji model knock-in
koji nosi najčešću mutaciju u ljudi, NPC1-I1061T, te je križan s modelom knock-out BACE1.
Neuropatološke značajke (uključujući gubitak Purkinjeovih neurona, poremećaj funkcije
endolizosomalnog puta, aktivaciju astrocita i mikroglija te hiperfosforilaciju proteina tau)
ispitane su u malom mozgu, kori velikog mozga i hipokampusu metodama
imunofluorescencije i Western blot. Delecija oba alela gena BACE1 u jedinki knock-in NPC1-
I1061T (N-KI) uzrokovala je ranu smrt uz povećanu progresiju svih neuropatoloških značajki
(izuzev odumiranja Purkinjeovih neurona) već u asimptomatskoj fazi bolesti. Delecijom
jednog alela gena BACE1 u jedinki N-KI ublaženo je nakupljanje ranih endosoma i lizosoma,
aktivacija astrocita te odumiranje Purkinjeovih neurona u simptomatskoj fazi bolesti.
Međutim, u završnoj fazi bolesti, delecijom jednog alela gena BACE1 u jedinki N-KI
povećana je aktivacija mikroglija te ubrzano odumiranje Purkinjeovih neurona, što je
uzrokovalo raniju smrt u odnosu na jedinke N-KI koje sadrže divlji tip gena BACE1.
Zaključno, genetičko smanjenje razine enzima BACE1 ima i povoljne i štetne učinke na
neuropatološke značajke bolesti NPC u mišjem modelu knock-in NPC1-I1061T, čime je
istaknuta važnost uloge enzima BACE1 i njegovih supstrata za funkciju mozga. Daljnja
istraživanja trebala bi razjasniti koji supstrati enzima BACE1 te koji tipovi stanica doprinose
patobiologiji bolesti NPC. Dobiveni rezultati doprinose boljem razumijevanju bolesti NPC,
kao i terapijskog potencijala enzima BACE1 u liječenju bolesti NPC i drugih
neurodegenerativnih poremećaja sličnog mehanizma nastanka
Abstract (english) Niemann-Pick type C disease (NPC) is a rare monogenic neurodegenerative disease and
lysosomal storage disorder. NPC is caused by mutation in NPC1 or NPC2 genes which
code for cholesterol transporters. Upon lack or dysfunction of NPC1 or NPC2 proteins,
free cholesterol accumulates in late endosomes and lysosomes causing disruption of the
endolysosomal pathway, profound neuroinflammation (activation of astrocytes and
microglia) and neurodegeneration of primarily Purkinje cells in the cerebellum.
Clinically, NPC is characterized by heterogenous phenotype including numerous
neurovisceral pathologies and early death. Despite etiological differences between NPC
and complex Alzheimer’s disease (AD), these disorders share several pathological
features, such as altered cholesterol metabolism, neurodegeneration, neuroinflammation
and accumulation of hyperphosphorylated tau and amyloid-β peptides in the brain.
Increased generation of amyloid-β due to increased BACE1-mediated proteolysis of APP
is considered a potential mechanism of AD pathogenesis. Interestingly, increased
BACE1-cleavage is observed in NPC as well. Since BACE1 is a potential
pharmacological target against AD, the goal of this doctoral thesis was to analyse the
effect of BACE1-genetic depletion on the levels and progression of neuropathological
features of NPC. Therefore, we used a knock-in murine model carrying the most common
human mutation, NPC1-I1061T, and crossed it with BACE1 knock-out mice.
Neuropathological features (including Purkinje cell loss, endolysosomal dysfunction,
activation of astrocytes and microglia, and hyperphosphorylation of tau) were analysed
in the cerebellum, cortex and hippocampus using immunofluorescence and Western blot.
Deletion of both BACE1 alleles in NPC1-I1061T knock-in (N-KI) mice caused early death
with increased progression of all neuropathological features analysed (except Purkinje
cell loss) already at the presymptomatic disease stage. Deletion of one BACE1 allele in
N-KI mice attenuated early endosome and lysosome accumulation, astrocyte activation,
and Purkinje cell loss at the symptomatic disease stage. However, at the terminal disease
stage, deletion of one BACE1 allele in N-KI mice enhanced microglial activation and
Purkinje cell loss, resulting in earlier death compared to N-KI mice containing wild-type
BACE1 gene. In conclusion, BACE1-genetic depletion has both beneficial and
detrimental effects on neuropathological features of NPC in NPC1-I1061T knock-in
murine model, which highlights the importance of BACE1 and its substrates in brain
function. Future studies should aim to determine which BACE1 substrates and which cell
types contribute to NPC pathobiology. These findings contribute to a better understanding
of NPC disease and the potential of BACE1-targeted therapies against NPC and other
neurodegenerative disorders with similar underlying pathologies.
Keywords
NPC1
I1061T
BACE1
nakupljanje kolesterola
endolizosomalni put
neurodegeneracija
neuroinflamacija
hiperfosforilacija proteina tau
Keywords (english)
NPC1
I1061T
BACE1
cholesterol accumulation
endolysosomal pathway
neurodegeneration
neuroinflammation
tau hyperphosphorylation
Language croatian URN:NBN urn:nbn:hr:193:969597 Promotion 2024 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2024-06-14 09:22:02
