Understanding the molecular mechanisms of γ-secretase in familial Alzheimer’s disease and its structure-function using nanobodies

γ-Secretase is an aspartic protease involved in the processing of the amyloid precursor protein (APP). Proteolytic cleavage of APP by this enzyme results in the production of Abeta peptides, which accumulate in the brain of Alzheimer’s disease (AD) patients. The familial form (FAD) is caused by mutations in one of three following genes: PRESENILIN 1 (PSEN1), PRESENILIN 2 (PSEN2) or AMYLOID PRECURSOR PROTEIN (APP). Although FAD only represents less than 1% of all AD cases, it offers a model to study the mechanisms underlying the disease. About 200 pathogenic mutations have been found in PSEN1, the catalytic active subunit of γ-secretase. How these mutations cause FAD remains a heavily debated topic in the AD field We investigated how APP processing by γ-secretase is affected by particularly aggressive AD-linked PSEN mutations. The results of our studies show that a shift in Aβ profiles towards production of the longer Aβ43 characterizes this particular pathogenic PSEN variants (Veugelen et al, Neuron, 2016). Further kinetic studies of other AD-linked PSEN mutations confirmed that enhanced generation of aggregation-prone peptides is the common denominator in FAD (Szaruga et al, J Exp Med, 2015). γ-Secretase structure-function studies have been limited due to the highly dynamic and conformationally flexible nature of the enzyme. Several reports describe the use of Nanobodies (Nbs) as conformational probes to dissect and study the conformational landscape of dynamic proteins (Dmitriev, Lutsenko, and Muyldermans, J Biol Chem, 2016). Nbs are single-domain camelid antibody fragments, consisting of the variable domain of a heavy chain only antibody (VH). To facilitate structure-function studies of this protease, we generated (in parallel experiments) 71 different anti-γ-secretase Nanobody families. We are currently characterizing their effects on γ-secretase activity.Table of Contents ........................................................................................... 9 List of Abbreviations .................................................................................... 13 Summary....................................................................................................... 17 Samenvatting ................................................................................................ 21 1. Introduction .......................................................................................... 25 1.1. Alzheimer’s disease ...................................................................... 25 1.2. APP processing and Aβ production .............................................. 29 1.3. γ-Secretase .................................................................................... 33 1.3.1. Presenilin ........................................................................................ 35 1.3.2. Nicastrin ......................................................................................... 37 1.3.3. Anterior Pharynx Defective 1 ......................................................... 38 1.3.4. Presenilin-Enhancer 2 ..................................................................... 38 1.4. γ-Secretase heterogeneity and functional implications ................. 39 1.5. Dynamic structure of γ-secretase .................................................. 40 1.6. Potential AD drug targets ............................................................. 42 1.6.1. Targeting α- and β-secretase: shifting the initial APP cleavage ..... 42 1.6.2. Targeting γ-secretase and Aβ ......................................................... 45 1.7. Tools to study γ-secretase structure-function ............................... 47 2. Research objectives.............................................................................. 51 3. Qualitative changes in human γ-secretase underlie familial Alzheimer’s disease .......................................................................................................... 53 3.1. Introduction .................................................................................. 54 3.2. Materials and Methods ................................................................. 57 3.3. Results .......................................................................................... 60 3.4. Discussion ..................................................................................... 72 4. Familial Alzheimer’s disease mutations in Presenilin generate amyloidogenic Aβ peptide seeds .................................................................. 75 4.1. Introduction .................................................................................. 75 4.2. Materials and Methods ................................................................. 78 4.3. Results .......................................................................................... 80 4.4. Discussion ..................................................................................... 85 5. Development of panning and screening methods for the generation of anti-γ-secretase nanobodies .......................................................................... 89 5.1. Introduction .................................................................................. 89 5.2. Phage display ................................................................................ 92 5.3. Critical aspects of biopannings ..................................................... 93 5.4. Sources of antigen ........................................................................ 94 5.4.1. Recombinant protein ...................................................................... 94 5.4.2. Whole cells or Virus-like particles (VLPs)..................................... 95 5.4.3. Membrane extracts ......................................................................... 96 5.5. Strategies of antigen presentation ................................................. 96 5.5.1. Antigen presentation on solid phase ............................................... 96 5.5.2. Antigen presentation in solution ..................................................... 97 5.5.3. Methods to capture γ-secretase ....................................................... 97 5.6. Methods of elution ........................................................................ 99 5.7. Screening strategies .................................................................... 100 5.7.1. Screenings for binders .................................................................. 101 5.7.2. Functional Screenings .................................................................. 107 5.8. Characterization of nbs ............................................................... 109 5.8.1. Defining the epitope ..................................................................... 110 5.8.2. Affinity determination .................................................................. 113 6. Generation and characterisation of nanobodies targeting γ-secretase 115 6.1. Introduction ................................................................................ 115 6.2. Materials and Methods ............................................................... 117 6.3. Results ........................................................................................ 120 6.4. Discussion ................................................................................... 127 7. Discussion .......................................................................................... 131 8. References .......................................................................................... 141 11 9. Acknowledgement, Personal Contribution and Conflict of Interest statements ................................................................................................... 175 10. Curriculum Vitae ............................................................................ 179nrpages: 179status: publishe

Familial Alzheimer's Disease Mutations in Presenilin Generate Amyloidogenic Ab Peptide Seeds

Recently it was proposed that the familial Alzheimer's disease (FAD) causing presenilin (PSEN) mutations PSEN1-L435F and PSEN1-C410Y do not support the generation of Aβ-peptides from the amyloid precursor protein (APP). This challenges the amyloid hypothesis and disagrees with previous work showing that PSEN1 FAD causing mutations generate invariably long Aβ and seed amyloid. We contrast here the proteolytic activities of these mutant PSEN alleles with the complete loss-of-function PSEN1-D257A allele. We find residual carboxy- and endo-peptidase γ-secretase activities, similar to the formerly characterized PSEN1-R278I. We conclude that the PSEN1-L435F and -C410Y mutations are extreme examples of the previously proposed "dysfunction" of γ-secretase that characterizes FAD-associated PSEN. This Matters Arising paper is in response to Xia et al. (2015), published in Neuron. See also the response by Xia et al. (2016), published in this issue.publisher: Elsevier articletitle: Familial Alzheimer’s Disease Mutations in Presenilin Generate Amyloidogenic Aβ Peptide Seeds journaltitle: Neuron articlelink: http://dx.doi.org/10.1016/j.neuron.2016.03.010 associatedlink: http://dx.doi.org/10.1016/j.neuron.2016.03.009 associatedlink: http://dx.doi.org/10.1016/j.neuron.2015.02.010 content_type: article copyright: © 2016 Elsevier Inc.status: publishe

The dynamic conformational landscape of γ-secretase

The structure and function of the γ-secretase proteases are of vast interest because of their critical roles in cellular and disease processes. We established a novel purification protocol for γ-secretase complex that involves a conformation and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase structure and its conformational variability. Under steady state conditions the complex adopts three major conformations, which are different in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors differentially stabilize sub-populations of particles with compact conformations, whereas a Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic population of inter-converting conformations, involving rearrangements at the nanometer scale and high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect Aβ generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely instrumental for highly allosteric behavior of the enzyme.status: publishe

Deletion of exons 9 and 10 of the Presenilin 1 gene in a patient with Early-onset Alzheimer Disease generates longer amyloid seeds

Presenilin 1 (PSEN1) mutations are the main cause of autosomal dominant Early-onset Alzheimer Disease (EOAD). Among them, deletions of exon 9 have been reported to be associated with a phenotype of spastic paraparesis. Using exome data from a large sample of 522 EOAD cases and 584 controls to search for genomic copy-number variations (CNVs), we report here a novel partial, in-frame deletion of PSEN1, removing both exons 9 and 10. The patient presented with memory impairment associated with spastic paraparesis, both starting from the age of 56years. He presented a positive family history of EOAD. We performed functional analysis to elucidate the impact of this novel deletion on PSEN1 activity as part of the γ-secretase complex. The deletion does not affect the assembly of a mature protease complex but has an extreme impact on its global endopeptidase activity. The mutant carboxypeptidase-like activity is also strongly impaired and the deleterious mutant effect leads to an incomplete digestion of long Aβ peptides and enhances the production of Aβ43, which has been shown to be potently amyloidogenic and neurotoxic in vivo.status: publishe

Qualitative changes in human γ-secretase underlie familial Alzheimer's disease

Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations.status: publishe

Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aβ peptides. The shift in Aβ length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aβ peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aβ. In contrast, E-Aβn stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aβ production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.status: publishe

Qualitative changes in human γ-secretase underlie familial Alzheimer’s disease

Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations