An integrative approach to elucidate the mechanisms and dynamics of Huntingtin aggregation and inclusion formation in neuronal models of Huntington's Disease

Despite the fact that the gene responsible for Huntington's disease (HD) is known, we still do not understand the underlying mechanisms leading to neurodegeneration and death. Identifying and understanding the mechanisms controlling mutant huntingtin (mHtt) aggregation and inclusion formation using different cellular and animal models is crucial to elucidate the molecular mechanisms underpinning the disease and to develop effective treatments to prevent or slow the progression of HD. At the mechanistic level, our work shows that mHtt aggregation and inclusion formation in the cytosol and nucleus occur via different mechanisms and lead to the formation of inclusions with distinct biochemical and ultrastructural properties. We show that mHtt cytoplasmic inclusion formation occurs via two phases: 1) a first phase involves the rapid formation of a dense fibrillar core and is driven predominantly by intermolecular interactions involving the polyQ domain via phase separation-like mechanisms; 2) a second phase is associated with the recruitment of soluble mHtt, fibril growth, and the active recruitment and sequestration of lipids, proteins, and membranous organelles. Our work points to this second phase as an important contributor to mHtt toxicity and suggests that targeting inclusion growth and maturation represent a promising therapeutic strategy. These observations suggest that the two types of inclusions may exert their toxicity via different mechanisms and may require different strategies to interfere with their formation, maturation and toxicity. Using primary neurons, we demonstrated that neuronal intranuclear inclusions evolve over time from small aggregates to large granulo-filamentous inclusions associated with cellular toxicity. In addition, our work underscores the failure of the cellular degradation machineries to clear mHtt inclusions, as well as the sequestration of other proteins, lipids and organelles. Finally, we would like to emphasize that our comparative analysis of tag-free and GFP-tagged mutant mHtt aggregation and inclusions formation will inform future efforts to develop models that reproduce HD pathology more faithfully and underscore the need for developing label-free techniques to investigate disease-relevant mechanisms that underpin inclusion formation.

Nucleation Dependent Polymerization of Amyloid-ß (Aß) as an Important Determinant of Aß Amyloid Formation and Neurotoxicity

Mohammad Asad Jan Qureshi

Aggregation and fibril formation of amyloid-β (Aβ) peptides play a pivotal role in the pathogenesis of Alzheimer's disease (AD). Aβ peptides, principally comprising of 40 or 42 amino acid residues (Aβ40 and Aβ42), are produced by proteolytic processing of the amyloid precursor protein by β and γ-secretase activity. A vast number of academic and biomedical industry projects are devoted to the discovery and development of therapeutic agents which inhibit the process of Aβ aggregation, and associated neurotoxicity, as potential disease modifying therapies for AD. Despite decades of research, the mechanism of Aβ neurotoxicity leading to development of AD and the identity of toxic Aβ species remain debatable. Nevertheless, independent lines of evidence implicate Aβ oligomers, which are precursors to fibrils, as putative toxic species and promising therapeutic targets. Aβ oligomers are characterized by heterogeneity in size and morphology distribution and have been shown to alter normal neuronal physiology in a manner that implicates their causal association with AD pathogenesis. The primary objective of this thesis work was to elucidate the structural determinants of Aβ neurotoxicity, the relationship of fibril formation process with neurotoxicity and identify neurotoxic Aβ species. Towards achieving these goals, we first developed and optimized reproducible protocols for generating various Aβ species, including monomers, protofibrils and fibrils, and established methods for detailed characterization of their structural properties. To better understand the relationship between Aβ aggregation and neurotoxicity, cell culture assays using primary neurons and neuronal cell lines were developed and used to assess the toxicity of various Aβ aggregates. Using these assays and biochemical tools, we sought to answer some of the key outstanding questions in the field concerning the role of Aβ amyloid formation process in neurodegeneration in AD. What is the nature of neurotoxic Aβ species and how does the process of its formation relate to neurodegeneration in AD? The identification of toxic Aβ species and/or process of its formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in AD, and development of effective diagnostic tools and therapeutic interventions. To achieve this, we successfully isolated protofibrils of distinct sizes and morphology distribution and compared their fibrillization and neurotoxicity to monomeric and fibrillar forms of Aβ. The results from our studies implicate an ongoing Aβ polymerization process, rather than distinct Aβ aggregate states (fibrillar or non-fibrillar), as primary determinant of Aβ neurotoxicity. We showed that crude Aβ42 preparations containing mixtures of monomers and heterogeneous oligomers were significantly more toxic than purified Aβ42 protofibril preparations. Subfractionation of protofibril preparations, to separate different protofibril species, resulted in further attenuation of their toxicity. The slow fibrillization of purified and subfractionated Aβ42 protofibrils was strongly linked to their diminished toxicity; thus, demonstrating that Aβ42 toxicity is not necessarily linked to specific prefibrillar aggregate(s), but rather to the ability of these species to grow and undergo fibril formation in presence of monomeric Aβ42. Consistent with this hypothesis, reintroduction of monomeric Aβ42 into these fractions restored amyloid formation and enhanced their toxicity to comparable levels as observed for the crude preparations. Furthermore, selective removal of monomeric Aβ42 from these preparations, using insulin degrading enzyme (IDE), reversed the toxicity of Aβ42 protofibrils. Together, these findings provide strong evidence thatAβ toxicity is linked to an ongoing Aβ polymerization process and is greatly reduced when the polymerization process is slowed down by selective removal/degradation of monomers. What is the molecular basis underlying the protective role of Aβ40 against Aβ42 amyloid formation and neurotoxicity in AD? The ratio of Aβ40/Aβ42 has been shown to be a critical determinant of neurodegeneration and the distribution of Aβ amyloid pathology in brain (parenchyma vs. vasculature) in AD. To investigate the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Aβ40/Aβ42, we assessed fibrillization in the mixtures of different species (monomers, protofibrils and fibrils) of the two peptides at wide range of ratios and concentrations. We showed that monomeric Aβ40 alters the kinetic stability, solubility, and morphological properties of Aβ42 aggregates and prevents their conversion into mature fibrils.Aβ40, at approximately equimolar ratios (Aβ40/Aβ42∼ 0.5-1), inhibits (>50%) fibril formation by monomeric Aβ42, whereas inhibition of protofibrillar Aβ42 fibrillogenesis is achieved at lower, substoichiometric ratios (Aβ40/Aβ42 ∼ 0.1). Additionally, we demonstrated that monomeric Aβ42 and Aβ40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Aβ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Aβ42 protofibrils selectively seed the fibrillogenesis of monomeric Aβ42 but not monomeric Aβ40. Finally, we also showed that the amyloidogenic propensities of different individual and mixed Aβ species correlate with their relative neuronal toxicities. Together, our results demonstrate that maintaining a delicate balance not only between the monomeric forms of Aβ42 and Aβ40, but also the monomeric and aggregated forms of both peptides is essential for maintaining Aβ solubility and preventing Aβ fibrillogenesis and toxicity. Implications for AD pathogenesis and anti-amyloid therapeutics This results presented in this thesis provide novel insight into the mechanisms of Aβ aggregation, fibrillogenesis and toxicity, and have important implications for understanding the role of Aβ amyloid formation in AD pathogenesis and design of therapeutic strategies. Most importantly, our results linking Aβ toxicity to the growth and fibrillization of Aβ oligomers in presence of Aβ monomers, suggest that anti-Aβ therapeutic strategies, many of which are currently being tested in animal models or in clinical trials in AD patients, hold potential as promising disease modifying interventions. We contemplate that targeting the nucleated polymerization of amyloid forming proteins offers an exciting framework for development of anti-amyloid therapies for a host of neurodegenerative diseases characterized by pathological accumulation of aggregated proteins including AD, Parkinson's disease, and prion diseases.

EPFL2010

Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer's disease research

Mohammad Asad Jan Qureshi, Hilal Lashuel

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-beta (Abeta) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Abeta aggregation contributes to neurodegeneration in AD and to the identity of the toxic Abeta species. In this paper, we describe methods and detailed protocols for reproducibly preparing Abeta aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Abeta aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Abeta aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used.

Nature Publishing Group2010

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease.