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Molekularna povezanost između homeostaze kolesterola, membranskog transporta i Alzheimerove bolesti Glavni istraživač Aberrant proteolytic cleavage of the amyloid precursor protein (APP) leading to increased formation/accumulation of b-amyloid peptide (AB) is considered a central event in the pahogenesis of Alzheimer's disease (AD). Although much has been known about the AB generation and its clearance, we still do not understand in great detail how AB metabolism is altered in the most common late-onset form of AD (LOAD) and what are the molecular trigger(s) that initiate these events. In this project we will test the hypothesis that cholesterol metabolism and membrane trafficking are tightly linked and that their dysregulation leads to Alzheimer's disease. AB is generated within the amyloidogenic beta-secretase pathway through the cascade of cleavage events involving APP processing by proteases B- (BACE1) and Y-secretases. All three proteins are transmembrane proteins, suggesting that lipid enviroment within the membrane and membrane trafficking could modulate their function and, thus, Ab formation. Indeed, B-secretase cleavage of APP mainly occurs within the endocytic pathway, while retrieval of APP from endosomes to trans-Golgi-network via retromers seems protective by depleting APP from BACE1. Dysfunction of endosomes and retromers is an early pathological feature in AD, supporting that these two membrane trafficking routes play an important role in the pathogenesis of AD. In addition to AB, B-secretase APP processing pathway generates C-terminal APP fragments (CTFB) which have previously shown to mediate endosome dysfunction (Jiang et al., 2010) and are responsible for memory impairment and neurotoxicity characteristic for AD (Neve et al., 1996). Substantial amount of evidence has shown that altered neuronal membrane cholesterol level and/or subcellular distribution is implicated in aberrant APP processing and AB/CTFB formation. However, the results are somewhat contradictory and we still do not have a complete understanding on how cholesterol can influence AD pathogenesis. In this project we will use a rare inherited cholesterol transport disorder Niemann-Pick type C (NPC), often referred as „childhood Alzheimer's“, to provide further evidence for the connection between APP metabolism, membrane misstrafficking and cholesterol homeostasis. Our approach is innovative as it utilizes an inherited single gene disorder to underpin the mechanism of the complex neurodegenerative diseases, such as AD. We will further examine the mechanistic details of an Alzheimer’s-like phenotype in NPC disease. In addition to AB peptide, which has been considered a central molecule in AD, in this project we will analyze how an increase in CTFB contributes to AD and NPC pathogenesis. We speculate that increased levels of CTFB, such as in AD and in NPC disease, regulate cholesterol homeostasis and modulate membrane trafficking within endocytic and/or retromer pathway, and, thus, contribute to the pathogenesis of AD and NPC disease. We will test the hypothesis that accumulation of APP-CTFB, at least in part, responsible for membrane misstrafficking and altered cholesterol homeostasis in NPC disease. We will analyze whether Alzheimer's-like phenotype in NPC disease is due to retromer dysfunction, as observed in LOAD. In line with the recent finding that CTFB binds cholesterol (Barrett et al., 2012) and that APP can control cholesterol homeostasis and turnover (Pierrot et al., 2013), we will analyze the role of accumulation of CTFB on endosome/retromer pathway and cholesterol homeostasis, as well as the role of its G700XXXG704 cholesterol binding motif in this process. We believe that these studies will identify CTFB as a signal transduction molecule involved in regulating cholesterol homeostasis and/or membrane trafficking and will provide clues on molecular targets for development of novel therapies against this devastating disorder. Ostali suradnici Prof. Lawrence Rajendran Co-Director Systems & Cell Biology of Neurodegeneration Division of Psychiatry Research University of Zurich Dr. Aleksandra Mladenović-Đorđević Department of neurobiology Institute for Biological Research “Siniša Stanković” Beograd, Serbia
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Malnar, Martina (2012) The mechanism(s) of cholesterol-effect on processing and transport of APP protein. Doctoral thesis, Sveučilište u Zagrebu, Prirodoslovno-matematički fakultet. Abstract Peptid amiloid-B (AB), središnja molekula u patogenezi Alzheimerove bolesti, nastaje amiloidogenim cijepanjem proteina APP B-sekretazom u endosomima, dok cijepanje proteina APP na staničnoj membrani a-sekretazom sprečava nastanak ovog patološkog peptida. Nakupljanje peptida AB u mozgu otkriveno je ne samo u Alzheimerovoj bolesti nego i u bolesti Niemann-Pick tipa C (NPC), nasljednoj neurodegenerativnoj bolesti nakupljanja lipida. U ovom radu smo pokazali da je povećana razina peptida AB u stanicama NPC posljedica povećanog cijepanja proteina APP enzimom B-sekretaza uzrokovanog nakupljanjem kolesterola. Nadalje, pokazali smo da je internalizacija proteina APP nužna za nastanak peptida AB, te da u stanicama NPC zbog nakupljanja kolesterola u kasnim endosomima / lizosomima dolazi do poremećaja u transportu proteina APP u endocitoznom putu. Naime, u stanicama NPC smo detektirali ubrzanu internalizaciju i usporeno recikliranje proteina APP na površinu stanice što uzrokuje njegovo nakupljanje u ranim / reciklirajućim endosomima, centralnom mjestu stvaranja peptida AB. Ovim istraživanjem smo pokazali da povišena razina kolesterola može biti pokretač patoloških procesa u Alzheimerovoj bolesti jer potiče smještaj proteina APP u endosomima što rezultira njegovim povećanim amiloidogenim cijepanjem i nastankom peptida AB.