«Submitted to the Department of Biology, Chemistry, and Pharmacy At the Freie Universität Berlin By Philipp Jäger From Hamburg, Germany February ...»
The Role of Autophagy in
Alzheimer Disease - from Cellular
Mechanisms to Systems Proteomics
To obtain the academic degree
Doctor rerum naturalium (Dr. rer. nat.)
Submitted to the
Department of Biology, Chemistry, and Pharmacy
At the Freie Universität Berlin
From Hamburg, Germany
Ex annis asperis, nunc propero ad Astra [sic].
This thesis work was performed between July 2006 and January 2011 under the
supervision of Prof. Dr. Tony Wyss-Coray at Stanford University, Palo Alto, CA, USA.
1st reviewer: Prof. Dr. Tony Wyss-Coray 2nd reviewer: Prof. Dr. Gerd Multhaup Disputation on the 23rd of May 2011.
Publications and statement of contribution Publications and statement of contribution This thesis has been submitted as a cumulative dissertation (Promotionsordnung des Fachbereiches Biologie, Chemie, Pharmazie der Freien Universität Berlin vom 04.09.2007 inkl. Erg. vom 07.02.2008). It is based on the following journal articles, both published and in preparation.
Title Contribution Jaeger PA, Britschgi M, Rufibach K, Burkholder B, Johns H, Sun 70% CH, Pradhan S, Petersen R, Knopman DS, Boeve BF, Boxer AL, Karydas A, Miller BL, Rademakers R, Dickson DW, Yonnkin S, Graff-Radford N, Wyss-Coray T. Plasma Protein Changes in Sporadic Alzheimer Disease Patients are Linked to Cognitive Decline and Identify Disease-related Pathways. (in preparation) Jaeger PA, Wyss-Coray T. Beclin 1 complex in autophagy and 100% Alzheimer disease. Arch Neurol. 2010 Oct;67(10):1181-4.
Jaeger PA, Pickford F, Sun CH, Lucin KM, Masliah E, Wyss-Coray 80% T. Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One. 2010 Jun 15;5(6):e11102.
Jaeger PA, Wyss-Coray T. All-you-can-eat: autophagy in 100% neurodegeneration and neuroprotection. Mol Neurodegener. 2009 Apr 6;4:16.
I confirm that the above stated author contributions are correct and that all my research has been conducted independently, following the ethical and practical standards of good scientific practice.
Philipp Jäger Palo Alto, the 25th of February 2011 Presentations Talks and poster presentations The author has presented the content of this research thesis at the following conferences.
PA. Jaeger, M. Britschgi, CH. Sun, H. Johns, S. Pradhan, RC. Petersen, DS. Knopman, BF. Boeve, AL. Boxer, A. Karydas, BL. Miller, R. Rademakers, DW. Dickson, N.
Graff-Radford, T. Wyss-Coray: TheCommunicome of a Disease: Application and Potential of Human Plasma Proteomics to Study Frontotemporal Dementia. 40th Annual Meeting of the Society for Neuroscience 2010, San Diego/CA, USA, Nov 13-17, 2010.
PA. Jaeger, M. Britschgi, H. Johns, CH. Sun, T. Wyss-Coray: Exploring the Communicome of Frontotemporal Dementia Patients through Plasma Proteomics.
Consortium for Frontotemporal Dementia Research (CFR): Research in Progress Meeting, The J. David Gladstone Institute, San Francisco/CA, USA, Jun 4, 2010.
PA. Jaeger, F. Pickford, CH. Sun, KM. Lucin, T. Wyss-Coray: Beclin 1 Regulates APP Turnover in the Endosomal-lysosomal Pathway. Cold Spring Harbor Meeting "Neurodegenerative Diseases", Cold Spring Harbor Laboratory, Cold Spring Harbor/NY, USA, Dec 4-7, 2008.
F. Pickford, PA. Jaeger, E. Masliah, T. Wyss-Coray: Amyloid Precursor Protein Processing, Autophagy, and Alzheimer’s Disease. Herbsttagung der Gesellschaft für Biochemie und Molekularbiologie (GBM), Hamburg, Germany, Sep 16-19, 2007.
PA. Jaeger, M. Britschgi, CH. Sun, S. Pradhan, H. Johns, T. Wyss-Coray: The Plasma Proteome of Secreted Cellular Communication Factors as a Tool to Study Dementia.
Keystone Symposium "Alzheimer's Disease Beyond Abeta", Copper Mt, CO, Jan 10-15, 2010.
PA. Jaeger, F. Pickford, CH. Sun, KM. Lucin, AR. LaSpada, T. Wyss-Coray: Beclin 1 Reduction Causes Accumulation of APP and APP Metabolites in Cells. Alzheimer’s Association Research Symposium, San Francisco/CA, USA, Jun 25, 2008.
F. Pickford, PA. Jaeger, E. Masliah, CH. Sun, M. Britschgi, S. Small, B. Spencer, E.
Rockenstein, N. Mizushima, B. Levine, T. Wyss-Coray: Effects of Autophagy on the Distribution and Processing of APP. 37th Annual Meeting of the Society for Neuroscience, San Diego/CA, USA, Nov 3-7, 2007.
F. Pickford, PA. Jaeger, E. Masliah, CH. Sun, M. Britschgi, S. Small, E. Rockenstein, N. Mizushima, B. Levine, T. Wyss-Coray: Beclin 1 Deficiency in Alzheimer's Disease Links Autophagy with Amyloidosis and Neurodegeneration. Keystone Symposium “Autophagy in Health and Disease”, Monterey/CA, USA, Apr 15-20, 2007.
Acknowledgements Acknowledgements This thesis is the cumulative work of many special people, united by the quest for scientific knowledge and medical improvements. First and foremost I would like to thank the patients and their families for allowing us to use their tissue samples for our research.
None of this would have been possible without their generosity and commitment.
Similarly important are our various funding sources, such as the National Institute of Health, the Department of Veterans Affairs, the John Douglas French Foundation, the Alzheimer Association, and the Consortium for Fronto-temporal Dementia Research.
I would also like to especially thank the Department of Biology, Chemistry, and Pharmacy at the Freie Universität Berlin, and specifically Prof. Dr. Gerd Multhaup, for showing a great deal of flexibility by supporting me to conduct my research on the other side of the world. The same is true for Stanford University, which has been an extraordinary place to work and study, and which has welcomed and nourished me with open arms. There, my special thanks go to the Graduate Program for the Neurosciences and the Stanford Institute for Neuro-Innovation & Translational Neurosciences, and their respective Directors and Administrators, past and present: Prof. Dr. William Newsome, Prof. Dr. John Huguenard, and Ross Colvin. They allowed me to participate in all program activities, both scientific and social, and made me immediately feel at home.
The laboratory of Prof. Dr. Tony Wyss-Coray has been a wonderful and extremely stimulating place to work, study, and develop a strong sense for the ways of scientific research. Many laboratory members have contributed to this work, both scientifically and spiritually, and I will not be able to name them all here. I would like to thank Dr. Fiona Pickford for helping me getting started in the laboratory as the first (!) graduate student, Dr. Markus Britschgi for sharpening my senses for details and precision, priceless when working with high-throughput data, and Dr. Hudson Johns, who helped me with countless hours of array scans and antibody and sample management.
Above all, I would like to express my deepest gratitude to Tony, not only for accepting me into his laboratory and generously funding my five years of research, but also for his fantastic and continuous support as a mentor, counselor, guide, colleague, and friend.
A vast amount of support came from my parents, Ruthild and Dr. Eckhard Jäger, who never rebelled against or vetoed my expatriate ambitions, always supported me with their unconditional love, and provided occasional (but highly appreciated) financial infusions. They joyfully embraced even my decision to marry an American woman, knowingly committing to many more years of long distance family reunions. They raised me to be who I am today, always encouraging my ventures to leave and see the world and to accept the foreign and strange not as an obstacle, but as a gem to be discovered. I promise to work hard to help them never feel the geographic distance!
Finally, my biggest Thank You of all goes to my charming and beautiful wife, Dr.
Lora Beatrice Jäger Sweeney. She supported me with all her energy throughout the last four years, supplied me with endless joy and happiness, challenged me with stimulating scientific discussions, and introduced me to the magic world of fly genetics and neurodevelopment. She is just the perfect mixture of a friendly competitor and a welcome detractor, always both pushing me to do my best at the bench and at the same time requesting my presence at terrible events such as skiing the Sierra Nevada Mountains, sailing the Pacific Ocean, hiking the Yellowstone National Park, or planting acres of tomato seedlings. And now she is the mother of our daughter. Nothing could be without her.
Table of contents Table of contents Publications and statement of contribution
Talks and poster presentations
Table of contents
List of figures
List of tables
Mechanism of autophagy
Beclin 1 in autophagy
Autophagy in neurodegenerative disease
Beclin 1 and autophagy in Alzheimer Disease
References for thesis introduction
Chapter 1: Autophagy in Neurodegeneration and Neuroprotection
Types of neuronal autophagy
Autophagy in the healthy nervous system
Autophagy as a clearing mechanism for protein degradation
Autophagy in vesicle sorting and organelle turnover
Autophagosomes as transport vacuoles
Regulation of autophagy
Autophagy in CNS disease and injury
Autophagy in chronic CNS diseases
Autophagy in acute CNS diseases and injuries
Autophagy and apoptosis
Table of contents
Chapter 2: Regulation of Amyloid Precursor Protein Processing by the Beclin 1 Complex
Activation of autophagy promotes APP, APP-CTF, and A! degradation
Becn1 knockdown increases APP, APP-like proteins, APP-CTFs, and A!..................76 Overexpression of APP does not change Becn1 or Pik3c3 protein levels
Reduction of Becn1 impairs degradation of autophagosomes and reduces Pik3c3 levels
Inhibition of autophagosome turnover leads to a reduction in Becn1 and Pik3c3 levels
Becn1 overexpression reduces APP immunoreactivity
AD brains have less BECN1 and PIK3C3 and more LC3
Material and methods
Chapter 3: The Beclin 1 Complex in Autophagy and Alzheimer Disease................108 Summary
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
Chapter 4: Plasma Protein Changes in Sporadic Alzheimer Disease Patients are Linked to Cognitive Decline and Identify Disease-related Pathways
Antibody microarrays can reliably measure relative protein levels in plasma............123
Table of contents
Secreted signaling protein levels in AD patients differ from non-demented control patients and from patients with non-AD dementia
Penalized linear regression modeling confirms and expands the pool of proteins-ofinterest
The connectivity between secreted signaling proteins increases significantly in AD patients
Meta-analysis of the different statistical modules to rank proteins-of-interest and correlation with an independent measure of cognitive decline
Protein-interaction, PubMed co-occurance, gene-ontology, miRNA target, and chromosome band analysis
TNF"-, TGF!-, and angiogenic signaling alterations in AD
Material and methods
Autophagy in Alzheimer Disease
Changes in systemic plasma factors and their effects on autophagy
References for thesis discussion
References for Chapters 1 to 4
List of figures List of figures Figure 1: APP trafficking and A! production
Figure 2: APP processing
Figure 3: Vesicle trafficking in autophagy
Figure 4: Beclin 1 deficiency in AD and APP transgenic mice
Figure 5: General thesis hypothesis
Figure 6: Steps in macroautophagy and chaperone mediated autophagy
Figure 7: Autophagy pathway in mammals
Figure 8: Control of autophagy
Figure 9: Interactions between autophagy and apoptosis
Figure 10: Expression of Becn1 and Pik3c3 in the mouse brain
Figure 11: Activation of autophagy promotes APP, APP-CTF, and A! degradation.......74 Figure 12: Effects of Atg5 knockdown on APP
Figure 13: Becn1 knockdown increases APP, APP-like proteins, APP-CTFs, and A!....78 Figure 14: Quantification of B103/hAPP cells
Figure 15: APP accumulation in CHO/hAPP cells after Becn1 siRNA
Figure 16: Effects of "-secretase inhibitors on Becn1 shRNA
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
Figure 19: Inhibition of autophagosomal turnover leads to a reduction in Becn1 and Pik3c3 levels
Figure 20: Pharmaceutical inhibition of autophagy in CHO/hAPP and B103/hAPP cells
Figure 21: Becn1 overexpression reduces APP immunoreactivity
Figure 22: Control experiments for Becn1 lentiviral overexpression
Figure 23: AD brains have less BECN1 and PIK3C3 and more LC3
Figure 24: Effects of BECN1 deficiency in AD
Figure 25: Autophagy in mammalian cells
Figure 26: The role of Beclin 1 in autophagy and Alzheimer Disease
List of figures
Figure 27: Experimental Design of the microarray production and analysis..................119 Figure 28: Antibody microarray performance
Figure 29: Plasma proteins with differential levels
Figure 30: Independent cohort confirmation
Figure 31: Plasma proteins with differential connectivity
Figure 32: Meta analysis of the plasma protein hits
Figure 33: Ingenuity Pathway Analysis and biological correlation analysis
Figure 34: Concept of the signaling factor array
List of tables List of tables Table 1: Presence of autophagy related gene expression in neuronal tissue