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Title: Morphological and molecular dissection of the gamma-secretase independent roles of Presenilins
Other Titles: Morphologische en moleculaire ontleding van de gamma-secretase onafhankelijke functies van Presenilines
Authors: Coen, Katrijn
Issue Date: 15-May-2012
Abstract: Alzheimer’s disease (AD) is characterized by a gradual lossof neurons and leads consequently to cognitive decline, concomitant with theformation of amyloid plaques and hyperphosphorylated Tau. The major constituentof these plaques is the neurotoxic peptide Aß, liberated from the amyloidprecursor protein (APP) by the consecutive actions of ß- and γ-secretase.Presenilins (PSEN1&2) form the catalytic components within the γ-secretasecomplex. Interestingly, most of the mutations in PSEN1, associated with early-onsetFAD cause the increased production of Aß42, which are more prone to aggregation(De Strooper, 2010a).PSENs perform also non-catalytic functions; several of themare still poorly understood, or it is still unsure whether or not theyrepresent bona fide non-γ-secretase associated functions. Studies illustratinga role for PSENs in protein maturation/turnover (Esselens et al., 2004;Hass et al., 2009; Khandelwal et al., 2007; Wilson et al., 2004) could reflect an underlyingmodulatory role of PSENs in endosomal transport. Finally, the fact thatendosomal transport blockades are observed in non-neuronal and neuronal cellsis moreover intriguing as endosomal/lysosomal dysfunction is amongst theearliest neuropathological feature observed in sporadic Alzheimer’s disease.Perturbations in these endosomal pathways may be one of the initiation steps inthe ethiology of AD, preceding the harmful effects of intracellular Aßaccumulations (Cataldo et al., 2004;LaFerla et al., 2007b).In order to decipher the contributions of γ-secretase-dependent/independentfunctions to AD, we first need to bridge the gap in our knowledge regarding theγ-secretase independent roles. In my doctoral thesis, I identified a major defect in aspecific endosomal transport and redistribution route in PSEN deficient cellsand hippocampal neurons. PSEN deficiency leads to the formation ofintracellular accumulations and causes delayed turnover of degradativeorganelles and late endosomes. Failure to perform proper recycling, as occursin PSENdKO cells, leads to impaired multivesicular body maturation andlysosomal function. A first major outcome of the thesis is that I demonstratedthat lysosomal dysfunction is not at all related to lysosomal acidificationproblems, as was proposed recently. A more detailed analysis moreoverdemonstrated that proton pump function and transport is normal in PSENdeficient cells. Secondly and in contrast, I show that lysosomal defects areindirectly related to a failure in selective endosomal routes, notably therecycling routes that is majorly regulated through the small GTPase ARF6.Endosomal recycling defects of lipid raft-associated and GPI-anchored proteins,but not their internalization, resulted in a selective depletion of thesemolecules from the cell surface of PSENdKO MEFs with serious implications oncell morphology andcell migration behavior. Moreover, we were able to fullyrescue all endosomal deficits by reintroducing the proteolytically inactivePSEN1 mutant or by normalizing the levels of the small GTPase ARF6. Our dataalso support that the failure in properly recycling endosomal cargo leads to abuild-up of e.g. cholesterol which could explain the observed defects inlysosomal calcium storage and therefore the impaired capacity of lysosomes tofuse and degrade long-lived proteins. This work implies that lateendosomal-lysosomal fusion deficits originating from a defect in lysosomalcalcium storage/release, MVB maturation and cell polarity/migration all fueldown to an ARF6-mediated recycling problem in PSEN deficient cells.An expression study in aging mice/neurons and control vs. ADbrain specimens revealed that PSENs and ARF6 were collectively downregulated.This implies that a defect observed in cell culture systems might indeed betranslated into an in vivo context, ultimately affecting the early-onset of ADneuropathology.Although, in general, it remains extremely difficult todissociate the different γ-secretase dependent and independent functions ofPSENs we now provide evidence that indeed non-catalytic functions of PSENsmight contribute to disease. We postulate that in sporadic cases,ARF6-deficiency might slow down endosomal recycling, resulting in increasedendosomal volume and decreased lysosomal clearance, exacerbating the toxicityof the intraneuronal Aß pool. The physiological outcome in PSEN deficientcells/neurons is likely the result of both γ-secretase dependent andindependent functions. PSENs may therefore act as a focal point influencing adiverse array of signaling molecules and protein trafficking routes that cancontribute to AD pathogenesis. By identifying a major endosomal sorting anddegradation pathway in PSEN deficient cells and hippocampal neurons, we herebypresent ARF6 as novel therapeutic target in the initiation cascade of ADpathology.
Table of Contents: TABLE OF CONTENTS
Dankwoord 3
Table of contents 5
List of abbreviations 8
Introduction 9
Intracellular transport regulation 9
A general overview 9
The secretory pathway 9
The endocytic system 11
Small GTPases in endocytic transport regulation 12
Clathrin-dependent endocytosis 13
Caveolin-dependent endocytosis 14
Clathrin- and Caveolin-independent endocytosis 15
The endosome machinery and its role in cell motility 17
Endosome to lysosome maturation 18
Lysosomal degradation 18
Macroautophagy 21
Alzheimer’s disease 23
The Amyloid cascade 23
APP and its secretases 23
The calcium hypothesis 25
Neurodegeneration and endo-lysosomal dysfunction 26
Presenilins and their versatile cellular functions 27
Protein transport and degradation 27
Cell adhesion and cell signaling 29
Calcium release from internal stores 30
Concluding remarks 31
Objectives of the research 32
Background and significance 32
General and Specific Aims 32
Materials & Methodology 34
Antibodies and Chemicals 34
Antibodies 34
Chemicals 34
Cell culture and primary hippocampal neuron culture 34
Cell culture 34
Primary hippocampal neuron culture 34
Retroviral and lentiviral transduction 34
Transfections 35
Cloning strategies 35
Cell Biology methods 37
Cell adhesion/migration experiments 37
Suspension-adhesion assay 37
Adhesion assay 37
Single cell migration 37
Wound healing analysis 38
Hippocampal neuron aggregation assay 38
Protein determination 38
Western blotting 38
Brain protein samples 38
Deglycosylation Experiments 38
Cell surface biotinylation 38
Semi‐permeabilization and in vitro COPII budding reaction 39
Analysis of small GTPases 39
ARF6 activity assay 39
Rac1/RhoA activity assays 39
Confocal microscopy 40
Pulse-chase of EGF-Alexa488 40
Analysis of lysosomes 40
Identification of acidic compartments in fixed samples 40
LysoTracker labeling and live imaging 40
DQ-BSA labeling 40
Cytosolic Ca2+ measurements 40
Lysosomal Ca2+ measurements 41
Lysosomal pH measurements 41
Genetic experiments 41
RNA isolation and RT-qPCR 41
Electroretinogram (ERG) recordings in Drosophila melanogaster 42
Microarray experiments 42
RNA isolation 42
Microarray Target Synthesis and Processing 42
Microarray data analysis 42
Proteomics 43
Synthesis of superparamagnetic nanoparticles 43
Isolation of the plasma membrane (PM) 43
Plasma membrane proteomics 43
Statistics 44
Chapter I: Endo-lysosomal dysfunction in PSEN-deficient cells is not caused by defects in the maturation and trafficking of the V-ATPase subunit V0a1 45
Abstract 45
Introduction 45
Results & Discussion 46
PSEN deficiency does not lead to defects in acidification 46
V0a1 subunit of the V H+-pump is not essential for lysosomal acidification 49
Post-golgi trafficking and N-glycosylation of endogenous and overexpressed V0a1 subunit were not affected in PSEN-/- 52
N-glycosylation deficient V0a1 subunit could still traffic to the plasma membrane 54
Mutation of a potential N-glycosylation site of Drosophila V0a1 subunit does not affect its function in vivo 58
Chapter II: Presenilins mediate endosomal recycling via ARF6 to maintain proper lysosomal clearance 60
Abstract 60
Introduction 60
Results 61
Presenilin deficiency affects the endocytic traffic of selected cargo molecules 61
The distinct morphology of PSENdKO MEFs reflects defects in migration/adhesion 65
Endo-lysosomal accumulations in PSENdKO MEFs originate from a deficiency in plasma membrane redistribution 67
ARF6 expression is down-regulated in PSEN deficient cells 74
PSEN gene deletion compromises lysosomal calcium homeostasis 75
ARF6 is a relevant target in aging and AD 78
Discussion 80
Global conclusions 84
Endo-Lysosomal and autophagic dysfunction in AD and other neurodegenerative disorders 84
AD genetics support the pathogenic importance of lysosomal proteolytic failure in AD 85
Autophagy in healthy neurons 87
Autophagy in AD is principally defective at the stage of autolysosomal proteolysis 87
Evidence for altered autophagy induction in AD is conflicting 89
Concluding remarks 93
Future perspectives 94
Summary 96
Samenvatting 98
List of references 100
Professional career 120
Education 120
Fellowships 120
Scientific publications 120
Poster publications and oral presentations 121
Patents 121
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Human Genetics - miscellaneous
Laboratory of Membrane Trafficking

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