Table of contents Publications and statement of contribution 3 Talks and poster presentations 4 Acknowledgements 6 Table of contents 8 List of figures 11 List of tables 13 Zusammenfassung (German) 14 Summary 16 Thesis Introduction 18 Alzheimer Disease 18 Mechanism of autophagy 23 Beclin 1 in autophagy 26 Autophagy in neurodegenerative disease 27 Beclin 1 and autophagy in Alzheimer Disease 29 Significance 31 References for thesis introduction 33 Chapter 1: Autophagy in Neurodegeneration and Neuroprotection 37 Summary 37 Background 37 Types of neuronal autophagy 38 Autophagy in the healthy nervous system 44 Autophagy as a clearing mechanism for protein degradation 48 Autophagy in vesicle sorting and organelle turnover 49 Autophagosomes as transport vacuoles 50 Regulation of autophagy 51 Autophagy in CNS disease and injury 55 Autophagy in chronic CNS diseases 55 Autophagy in acute CNS diseases and injuries 61 Autophagy and apoptosis 64 Concluding remarks 66 Abbreviations 66 Chapter 2: Regulation of Amyloid Precursor Protein Processing by the Beclin 1 Complex 68 Summary 68 Background 69 Results 73 Activation of autophagy promotes APP, APP-CTF, and Aβ degradation 73 Becn1 knockdown increases APP, APP-like proteins, APP-CTFs, and Aβ 76 Overexpression of APP does not change Becn1 or Pik3c3 protein levels 82 Reduction of Becn1 impairs degradation of autophagosomes and reduces Pik3c3 levels 84 Inhibition of autophagosome turnover leads to a reduction in Becn1 and Pik3c3 levels 86 Becn1 overexpression reduces APP immunoreactivity 91 AD brains have less BECN1 and PIK3C3 and more LC3 94 Discussion 98 Material and methods 103 Acknowledgements 107 Chapter 3: The Beclin 1 Complex in Autophagy and Alzheimer Disease 108 Summary 108 Background 108 Autophagy is a vesicular degradation pathway for cytosolic components 109 The Beclin 1 Connection: Autophagy, Neurodegeneration, and Alzheimer’s 112 Clinical Relevance and Current Research 115 Chapter 4: Plasma Protein Changes in Sporadic Alzheimer Disease Patients are Linked to Cognitive Decline and Identify Disease-related Pathways 117 Summary 117 Background 118 Antibody microarrays can reliably measure relative protein levels in plasma 123 Secreted signaling protein levels in AD patients differ from non-demented control patients and from patients with non-AD dementia 125 Penalized linear regression modeling confirms and expands the pool of proteins-of-interest 131 The connectivity between secreted signaling proteins increases significantly in AD patients 134 Meta-analysis of the different statistical modules to rank proteins-of-interest and correlation with an independent measure of cognitive decline 138 Protein-interaction, PubMed co-occurance, gene-ontology, miRNA target, and chromosome band analysis 142 TNFα-, TGFβ-, and angiogenic signaling alterations in AD 145 Discussion 148 Material and methods 151 Thesis Discussion 153 Autophagy in Alzheimer Disease 153 Changes in systemic plasma factors and their effects on autophagy 154 Outlook 157 References for thesis discussion 158 References for Chapters 1 to 4 159 Appendix: 180 Curriculum Vitae 181 Original Publications 185 List of figures Figure 1: APP trafficking and Aβ production 21 Figure 2: APP processing 22 Figure 3: Vesicle trafficking in autophagy 25 Figure 4: Beclin 1 deficiency in AD and APP transgenic mice 30 Figure 5: General thesis hypothesis 32 Figure 6: Steps in macroautophagy and chaperone mediated autophagy. 41 Figure 7: Autophagy pathway in mammals. 42 Figure 8: Control of autophagy 54 Figure 9: Interactions between autophagy and apoptosis 65 Figure 10: Expression of Becn1 and Pik3c3 in the mouse brain 72 Figure 11: Activation of autophagy promotes APP, APP-CTF, and Aβ degradation 74 Figure 12: Effects of Atg5 knockdown on APP 75 Figure 13: Becn1 knockdown increases APP, APP-like proteins, APP-CTFs, and Aβ 78 Figure 14: Quantification of B103/hAPP cells 79 Figure 15: APP accumulation in CHO/hAPP cells after Becn1 siRNA 80 Figure 16: Effects of γ-secretase inhibitors on Becn1 shRNA 81 Figure 17: Overexpression of APP does not change Becn1 or Pik3c3 protein levels 83 Figure 18: Reduction of Becn1 implairs degradation of autophagosomes and reduced Pik3c3 levels 85 Figure 19: Inhibition of autophagosomal turnover leads to a reduction in Becn1 and Pik3c3 levels 89 Figure 20: Pharmaceutical inhibition of autophagy in CHO/hAPP and B103/hAPP cells 90 Figure 21: Becn1 overexpression reduces APP immunoreactivity 92 Figure 22: Control experiments for Becn1 lentiviral overexpression 93 Figure 23: AD brains have less BECN1 and PIK3C3 and more LC3 96 Figure 24: Effects of BECN1 deficiency in AD 102 Figure 25: Autophagy in mammalian cells 111 Figure 26: The role of Beclin 1 in autophagy and Alzheimer Disease 114 Figure 27: Experimental Design of the microarray production and analysis 119 Figure 28: Antibody microarray performance 124 Figure 29: Plasma proteins with differential levels 129 Figure 30: Independent cohort confirmation 130 Figure 31: Plasma proteins with differential connectivity 136 Figure 32: Meta analysis of the plasma protein hits 140 Figure 33: Ingenuity Pathway Analysis and biological correlation analysis 147 Figure 34: Concept of the signaling factor array 156 List of tables Table 1: Presence of autophagy related gene expression in neuronal tissue 43 Table 2: Neuronal phenotype of autophagy related knockout/knockdown animals. 46 Table 3: Autophagy in common chronic neurodegenerative diseases 60 Table 4: Autophagy in acute neuronal injury 63 Table 5: Detection of autophagy proteins in human AD brain tissue 97 Table 6: FDA approved, autophagy inducing drugs 116 Table 7: Functional grouping of the secreted plasma proteins 122 Table 8: Human sample demographics 126 Table 9: Plasma proteins with differential levels 127 Table 10: eNet comparison and ranking 132 Table 11: Meta-analysis of the plasma proteins (experimental data, top 25 hits) 139 Table 12: Functional ontology analysis of the protein hits 144, I am interested in inflammation and protein aggregation in neurodegenerative diseases. I pursued three main questions during my PhD project in the Wyss- Coray lab: Does autophagy contribute to the metabolism of Amyloid Precursor Protein (APP) in the brain? Does autophagy activity play a role in Alzheimer Disease (AD) pathology? And how does inflammation and cellular communication influence dementia pathology in humans? My PhD project is based on the discovery that Beclin 1 (BECN1), a protein involved in autophagy initiation, is selectively reduced in AD patients' cortex. We created an AD mouse model with reduced BECN1 levels and observed significantly enhanced deposition of Aβ plaques, increased microglia activity, and increased neuronal loss (Pickford et al., 2008). Next, I established various cell culture models of BECN1 deficiency and overexpression (using siRNA and Lentivirus) and demonstrated that BECN1 regulates APP levels through autophagy. Accordingly, I found that enhancing autophagosomal turnover through starvation or pharmacological treatments reduced levels of APP and its metabolites (Jaeger et al., 2010). To explore how systemic inflammatory, immune signaling, and cellular communication factors modulate neuronal processes (and potentially autophagy) in neurological disorders we developed an antibody-based protein microarray technique to simultaneously measure hundreds of plasma based communication factors (chemokines, cytokines, growth factors, neurotrophins etc.) in blood from human dementia patients and unaffected controls. I adapted existing genomics tools and developed novel data extraction, data handling, and analytical methods to interpret the plasma proteomics data. I discovered a significant de-regulation of a variety of important biological pathways such as TNF-α or TGF-β signaling (Jaeger et al., manuscript in preparation). Based on my findings, our laboratory will now continue to explore potential candidate pathways that might underlie the observed de-regulation of brain autophagy in AD, both on a cellular and a systemic level., Während meiner Doktorarbeit lag der Schwerpunkt meines wissenschaftlichen Interesses bei der Erforschung schädlicher Protein-Aggregate und von Entzündungsstoffen, und deren Rolle bei der Entstehung neurodegenerativer Erkrankungen. Drei zentrale Fragen haben mich dabei beschäftigt: Spielt die Autophagie bei der Verstoffwechslung des Amyloid-Vorläufer Proteins (APP) im Gehirn eine Rolle? Hat die Aktivität der Autophagie einen Anteil an der Entstehung der Alzheimer Erkrankung? Und was für ein Zusammenhang besteht zwischen einer fortschreitenden, systemweiten Entzündungs-Reaktion, intrazellulärer Kommunikation und der Alzheimer Erkrankung im Menschen? Meine Doktoarbeit basiert auf der Entdeckung, dass Beclin 1 (BECN1), ein Protein, das eine wichtige Rolle bei der Initiation von Autophagie spielt, im Cortex von Alzheimer Patienten reduziert zu sein scheint. Unser Labor entwickelte daraufhin ein Alzheimer-Maus-Modell mit reduzierter BECN1 Expression und stellte fest, dass diese Mäuse unter erhöhter Ablagerung von Aβ Plaques, erhöhter Aktivität von Mikroglia und fortgeschrittenem Verlust von Nervenzellen leiden (Pickford et al., 2008). Um den Zusammenhang zwischen BECN1, Autophagie und Alzheimer Erkrankung besser zu verstehen, habe ich daraufhin eine Reihe verschiedener Zellkultur-Experimente entwickelt. Verringerung und Über-Exprimierung von BECN1 durch siRNA Plasmide und Lenti- Virus Partikel haben mir geholfen aufzuzeigen, dass der zelluläre APP Gehalt tatsächlich durch Autophagie reguliert werden kann. Dementsprechend kann APP gezielt abgebaut und eine Aβ-Ansammlung verringert werden, wenn man Autophagie entweder durch Nährstoffs-Entzug oder durch Pharmaka künstlich aktiviert (Jaeger et al., 2010). Um der Frage nachzugehen, wie Entzündungsstoffe, Immunsignale und zelluläre Kommunikations-Faktoren während einer neurodegenerativen Erkrankung die Vorgänge (u.U. auch die Autophagie) in Nervenzellen beeinflussen, hat unser Labor einen speziellen antikörper- basierten Biochip entwickelt. Mit diesem Biochip sind wir in der Lage, hunderte verschiedener Kommunikations-Faktoren (z.B. Chemokine, Zytokine, Wachstumsfaktoren, Neurotrophine usw.) in Blut-Plasma-Proben demenz-erkrankter Patienten oder gesunder Probanden zu messen. Basierend auf diesem Chip-Design habe ich neuartige Methoden zur Daten-Extraktion, Daten-Analyse und Daten- Interpretation entwickelt. Dabei habe ich eine signifikante Deregulation in verschiedenen Signal-Kaskaden entdeckt, wie zum Beispiel in der TNF-α oder der TGF-β Kaskade (Manuskript in Vorbereitung). Unser Labor ist nun damit beschäftigt, diese potenziellen Signal-Kaskaden weiter zu erforschen, und zu überprüfen, welche bei der beobachteten Reduktion der Autophagie beteiligt sein könnten.