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Structural characterization of amyloid beta oligomers with functional links associated to Alzheimer's disease

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Universitat de Barcelona. Facultat de Química

# metadata.dc.description.abstract[].value
[eng]  Alzheimer’s disease (AD) is the most common form of dementia. It was first described in 1906 by Alois Alzheimer. Later on, in 1984 George Glenner and Colin Masters isolated the amyloid-beta (Aβ) peptide from a human brain and associated it to the disease. Since then the amyloid hypothesis has been a rather controversial matter discussed among the scientific community. This is because although Aβ has been targeted by the majority of the drugs in clinical trials not even one has been approved up to date: 13 have been discontinued and 10 are in phase 3 clinical trials. A possible explanation for these devastating numbers is the high complexity of the target due to the variety of aggregation forms that Aβ can adopt. Therefore, understanding the links between protein aggregation and neurotoxicity, and specially obtaining the 3D structures of the aggregates responsible for neurotoxicity is key to design effective diagnostic and therapeutic strategies. Unfortunately, this remains one of the most important unresolved issues in the field. The group of Dr. Carulla has been working on the hypothesis that Aβ interacts with the cell membrane leading to ionic dyshomeostasis. In order to study this scenario, the group has changed the paradigm and treated Aβ as a membrane protein and applied well known methodologies used to characterize this family of proteins to study Aβ. By doing so, the group has proved that Aβ is able to form a type of oligomer in the presence of detergent micelles which adopts a very specific and defined structure with characteristics of a β-barrel assembly and functions as a pore. They refer to these types of oligomer as β-Barrel Pore-Forming Oligomer (βPFO). 
Hereby we present the work carried out to identify by using different biophysical techniques, the 3D structure of βPFO. As a starting point, we have used detergents to study the oligomerization process in a membrane mimetic environment. Micelles compared to other more native-like biomimetics environments based on lipids, will enable the application of novel mass spectrometry (MS) strategies and well-established solution NMR techniques thus providing high-resolution structural information. Since the accumulation of different amounts of Aβ in the membrane is a plausible scenario in the context of the disease, we have used different Aβ to detergent micelle ratios ([Aβ]:[M]) to study the role of this variable in the oligomerization process of Aβ. Throughout the work done we have optimized not only the ratio but also other conditions such as the buffer and the pH to modulate the preparation of samples enriched in defined oligomer populations. 
To study the stoichiometry of βPFO, we used with Native Mass Spectrometry which proved to be an adequate technique to preserve the non-covalent interactions of our samples analyse them in the gas phase. One of the key parts of the project consisted in the screening of a wide range of non-ionic detergents compatible with MS. After this work as we were able to identify Pentaethylene Glycol Monooctyl Ether (C8E5) as the best candidate for our samples. To continue working with the different samples we implemented a new approach based on coupling size exclusion chromatography (SEC) directly to a SYNAPT G2. This approach has allowed us to establish that higher molecular weight oligomers are better preserved and therefore better detected as we increase the signal to noise ratio.  This enabled us to study different points of the SEC chromatogram and therefore understand better the composition of our samples and our system. For the standard βPFO samples, we reported specific charge states for the octamer and tetramer species.
In parallel to complement the native-MS results, we have also worked to develop a method to analyse chemically cross-linked βPFOs by MALDI-MS. After a process of trials and optimizations we established a zero-length cross-linker (DMTMM) which allowed us to cross-link the βPFOs and detect again tetramer and octamer such as in the native-MS approach. 
In order to assess the relevance and to potentially validate the standard βPFO preparation as a target for AD’s it is crucial to characterize the binding of the Nanobodies to the oligomer. This work will also give us the opportunity to generate Nanobodies that could recognize their specific structures in brain tissue and thus assess whether the oligomers proposed are related to AD’s and if so, evaluate them as new targets for AD. Moreover we are very interested in the potential use of these Nanobodies as novel diagnostics or therapeutics tools.

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[cat]  La malaltia d'Alzheimer (AD) és la forma més comuna de demència. Va ser descrita per primera vegada el 1906 per Alois Alzheimer. Més endavant, al 1984, George Glenner i Colin Masters van aïllar el pèptid amiloide-beta (Aβ) d'un cervell humà i el van associar a la malaltia. Des de llavors, la hipòtesi amiloide ha estat un tema bastant controvertit discutit entre la comunitat científica. Una possible explicació és l’alta complexitat del sistema a causa de la varietat de formes d’agregació que Aβ pot adoptar. Per tant, entendre els vincles entre l'agregació de proteïnes i la neurotoxicitat, i especialment l'obtenció de les estructures 3D dels agregats responsables de la neurotoxicitat, és clau per dissenyar estratègies diagnòstiques i terapèutiques efectives. Malauradament, aquest tema continua sent un dels problemes pendents més importants. El grup de la Dra. Carulla ha estat treballant en la hipòtesi que l'Aβ interactua amb la membrana cel·lular que condueix a una deshomeostasi iònica. Per estudiar aquest escenari, el grup ha canviat el paradigma i ha tractat Aβ com a proteïna de membrana i aplicant tècniques biofísiques ben establertes per a caracteritzar proteïnes de membrana per tal d’estudiar Aβ. D'aquesta manera, el grup ha demostrat que Aβ és capaç de formar un tipus d'oligòmers en presència de micel·les de detergent que adopten una estructura molt específica i definida amb capacitat de formar porus a través de membranes lipídiques. Es refereixen a aquest tipus d’oligòmers com a oligòmers formadors de porus barril β (βPFO). A la present tesi doctoral, presentem l’estudi realitzat per identificar mitjançant diferents tècniques biofísiques, l'estructura 3D de βPFO. Hem utilitzat detergents per estudiar el procés d’oligomerització en un entorn mimètic de membrana. Les micel·les en comparació amb altres entorns biomimètics basats en lípids, permeten l'aplicació d’estratègies d'espectrometria de masses (MS) i de ressonància magnètica nuclear (RMN) ben establertes, proporcionant així informació estructural d'alta resolució. Atès que l’acumulació de diferents quantitats d’Aβ a la membrana és un escenari plausible en el context de la malaltia, hem utilitzat diferents relacions de micel·les Aβ a detergents ([Aβ]: [M]) per estudiar el paper d’aquesta variable en l’oligomerització. procés d'Aβ.

# metadata.dc.title[].value
Structural characterization of amyloid beta oligomers with functional links associated to Alzheimer's disease