La maladie d'Alzheimer (MA) est une maladie neurodégénérative dévastatrice qui touche un grand nombre de personnes. Elle entraîne des troubles de la mémoire et, éventuellement, une perte complète des fonctions cognitives. Le peptide amyloïde-β (Aβ) et la protéine associée aux microtubules tau sont généralement associés à la perte progressive de la mémoire. Dans les stades précoces, la MA se caractérise par une perturbation générale de l'efficacité synaptique. Les effets perturbateurs d'Aβ sur la plasticité à long terme sont bien documentés, par contre nos connaissances des effets immédiats du peptide sur la transmission synaptique sont limitées. Mon hypothèse est qu’Aβ ne nécessite pas une période prolongée pour promouvoir des changements de la transmission synaptique et qu’il peut modifier la fonction synaptique même après une exposition aiguë. Dans l’étude I, je test cette hypothèse à l’aide d’une exposition aiguë d'oligomères Aβ sur des tranches organotypiques d'hippocampe. Mes résultats indiquent qu’Aβ favorise une dépression synaptique sur les neurones pyramidaux hippocampiques avec une cinétique relativement rapide. Je démontre également que la dépression synaptique dépend de l'activation des récepteurs NMDA (NMDAR), mais ne dépend pas du flux d'ions à travers son canal ionique. Puisqu’il a été démontré que l'activation des NMDAR entraîne la phosphorylation de tau, il est plausible qu’Aβ modifie l'excitabilité des neurones en modulant la phosphorylation de cette protéine. Étant donné que les NMDAR jouent un rôle important dans la plasticité synaptique à long terme, cette chaîne d’événements moléculaires pourrait contribuer aux déficits de plasticité observés dans les phases initiales de la MA. Mon hypothèse est qu’Aβ modifie l’activité synaptique en modulant la phosphorylation de tau. Pour tester cette hypothèse, j’induis, dans des neurones de tranches de l’hippocampe, l’expression de formes de tau contenant des mutations qui bloquent la phosphorylation de la protéine aux sites ciblés. Dans l’étude II, j’observe que la phosphorylation de tau aux sites AT8 et AT180 régule l’expression de la plasticité synaptique ainsi que le dysfonctionnement de la transmission synaptique induits par les oligomères d’Aβ. Je démontre aussi que la phosphorylation du site PHF-1 ne contribue pas à la régulation de la plasticité et de la transmission synaptique. Puisque les sites AT8 et AT180 régulent l’interaction de la protéine tau avec la tyrosine kinase Fyn, mes résultats suggèrent que l’interaction entre tau et Fyn est importante pour l’expression de la plasticité synaptique et de la dépression favorisées par les oligomères d’Aβ. En effet, je démontre que l’inhibition de l’activité de la kinase Fyn résulte en un blocage de la dépression synaptique à long terme et un sauvetage de la fonction synaptique en présence d’Aβ. Je conclus que la phosphorylation de la protéine tau à des sites spécifiques est indispensable à l’expression de la plasticité synaptique et j’émets l’hypothèse que les oligomères d’Aβ modifient l'activité synaptique en influençant la stabilité du complexe Fyn-tau. Je propose donc que la perturbation de la stabilité de ce complexe peut être utilisée en thérapie pour inverser les déficits synaptiques dans les stades précoces de la MA. / Alzheimer disease is the most common form of dementia; it is characterized by problems in memory formation, which with time leads to a complete loss of cognitive functions. The peptide amyloid-β (Aβ) and the microtubule-associated protein tau are commonly believed to be responsible for the decline in memory formation. In the early stages of AD, this is thought to happen through a general disruption in synaptic efficiency. The disruptive effects of Aβ on long-term plasticity are well documented; however, little is known about the immediate effects of the peptide on synaptic transmission. My hypothesis is that Aβ does not need a prolonged period to promote changes in synaptic transmission, and that the peptide is able to affect synaptic function even after an acute exposure. In study I, I investigate this hypothesis using an acute exposure of Aβ oligomers to organotypic hippocampal slices. I report that Aβ promotes synaptic depression on hippocampal pyramidal neurons with a fairly rapid kinetic. I also show that the synaptic depression is dependent on the activation of the NMDAR, but independent on the ion flux through the channel. Because it was shown that the activation of the NMDAR leads to phosphorylation of tau, it appears feasible that Aβ modifies neuronal excitability by modulating tau phosphorylation. Since the NMDAR plays a critical role in the induction of long-term plasticity, this cascade of events could contribute to the deficits in plasticity observed during the initial stages of AD. My hypothesis is that Aβ modifies synaptic activity by modulating phosphorylation on tau. To test this hypothesis, I express in hippocampal neurons tau mutants in which phosphorylation on specific sites is blocked. In study II, I report that phosphorylation on tau at the AT8 and AT180 sites regulates the expression of synaptic plasticity as well as the dysfunction in synaptic transmission induced by Aβ oligomers. I also show that phosphorylation at the PHF-1 site is not involved in mediating either effects. Since the AT8 and AT180 sites regulate the interaction of tau protein with the tyrosine kinase Fyn, my results suggested that the interaction between tau and Fyn is important for the expression of synaptic plasticity and the depression mediated by Aβ oligomers. Indeed, I show that inhibiting the activity of Fyn kinase results in a block of LTD and a rescue of synaptic function in presence of Aβ. I conclude that phosphorylation of tau at specific sites is mandatory for the expression of synaptic plasticity, and suggest that Aβ oligomers promote changes of synaptic activity by influencing the stability of the tau-Fyn complex. I therefore propose that disrupting the stability of this complex can be exploited therapeutically to rescue synaptic deficits in the initial stages of AD.

Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.

As of today, Alzheimer’s disease is the leading cause of dementia among neurodegenerative disorders, affecting many millions of people worldwide. As the average life span of populations increase, more and more people succumb to the illness each year. Like other neurodegenerative disorders, Alzheimer’s disease can be attributed to the accumulation of protein aggregates in the brain. These amyloid-β peptides and tau proteins can presumably be detected in the brain many years before the onset of clinical symptoms. Development of fluorescent ligands, capable of binding to these neuropathological hallmarks and highlighting them, could serve as molecular diagnostic tools and facilitate an early diagnosis of the disease. The method could also be useful in studying disease progression and evaluating the effects of novel treatments. One such ligand is HS-259. The aim of this project was to synthetize different analogues of HS-259, and test their selectivity towards the aforementioned aggregates in brain tissue from an individual with Alzheimer’s disease. Staining of tissue samples with analogue solution enables visualization of aggregate sites through fluorescence imaging. In the end, five analogues were synthetized, albeit in relatively low overall yields. Synthetic methods included Suzuki-Miyara cross-couplings, Ullmann-type arylations and condensations. Liquid Chromatography-Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used for analysis of the compounds. Two of the five analogues could be tested for staining of aggregates and assessed for photophysical characteristics, i.e. absorption- and emission spectra. One analogue stained both amyloid-β aggregates and some tau aggregates, whereas the other stained neither. Since only two analogues were tested and rendered inconsistent results, further studies are needed to assess the binding properties of HS-259 analogues in general.

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