Alzheimer's disease (AD) is characterised by a series of biomolecular aggregation events, which include the formation of neurotoxic protein structures composed of the β-amyloid (Aβ) peptide. In this thesis, fluorescence self-quenching (FSQ) between fluorescently-labelled peptides is introduced as a strategy for detecting and characterizing Aβ aggregates in solution, and for overcoming limitations associated with conventional methods. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, the fluorescence response of HiLyte Fluor 555-labelled Aβ peptides is characterised to demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, N-terminal tagging of β-amyloid peptides is shown to not alter the self-assembly kinetics or the resulting aggregated structures. When performed in Förster resonance energy transfer (FRET) format, this method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid self-assembly. The ability of FSQ-based methods to monitor the inhibition of Aβ aggregation by model test compounds including the small heat shock protein (Hsp), the amyloid-binding alcohol dehydrogenase protein (ABAD) and bovine serum albumin (BSA) is also demonstrated. Given that Aβ is formed within the cell membrane and is known to induce its disruption, sophisticated single-molecule fluorescence spectroscopy methods were developed to quantify membrane dynamics induced by the presence of disrupting agents, such as Aβ and detergents. The solubilisation dynamics of single liposomes induced by the non-ionic surfactant Triton-X 100 (TX-100) were studied in real-time. Using this approach, the swelling and permeabilization steps of the solubilisation process were unambiguously separated within single FRET trajectories, and their kinetic details as a function of Triton-X 100 and presence of cholesterol within the membrane structure were examined. Finally, single-molecule stepwise-photobleaching techniques were employed to study the effect of Aβ oligomers interacting with supported-lipid bilayers, establishing a platform from which to investigate how the presence of a membrane layer affects Aβ oligomerization at the level of individual molecules. Overall, the fluorescence-based strategies for amyloid- and liposome-sensing presented in this work bridges the gap between current morphology-specific techniques and highly-specialized single-molecule methods to provide a biophysical toolbox to investigate the changes in structure, size and molecular interactions accompanying the amyloid aggregation pathway and for the screening of novel therapeutic and diagnostic agents.

La maladie d’Alzheimer (MA) se présente souvent comme un déclin progressif des fonctions cognitives et comportementales. Au niveau biologique, on observe une accumulation de la protéine bêta-amyloïde (Aß) et de la protéine tau, une altération de la fonction synaptique ainsi qu’une mort neuronale. Des travaux récents ont démontré qu’une souche spécifique de probiotiques, Lacticaseibacillus rhamnosus HA-114 (HA-114), avait des propriétés neuroprotectrices dans plusieurs modèles de maladies neurodégénératives. Le projet de recherche présent visait à caractériser les aspects neurophysiologiques d’une souche de nématodes transgéniques exprimant l’Aß et valider la neuroprotection de HA-114 dans ce modèle ainsi que dans un modèle murin de la MA (APP/PS1). Les résultats présentés dans ce manuscrit montrent que les vers transgéniques exprimant le fragment 1-42 de l’Aß récapitulent des caractéristiques clés de la MA, soit une altération de la santé neuronale et de la mémoire liée à l’âge. Il s’agit donc d’un nouvel outil pour l’investigation de la physiopathologie de la MA et pour l’essai de molécules ayant un potentiel thérapeutique. Les données expérimentales montrent aussi que l’administration de HA-114 permet d’atténuer les phénotypes de neurodégénérescence et de paralysie liée à l’âge. Cela témoigne d’un potentiel en vue de l’amélioration de la qualité de vie des patients qui souffrent de la MA. L’étude des effets de HA-114 dans un modèle murin pourrait révéler de nouvelles avenues d’investigations pour la physiopathologie de la MA et les mécanismes par lesquels on pourrait un jour la traiter. / Alzheimer’s disease (AD) often presents itself as a progressive decline of behavioral and cognitive functions such as memory impairment. On a biological level, this disease presents extracellular accumulation of amyloid-beta (Aß), intracellular accumulation of tau, alteration of synaptic function and neuronal loss. Recent data showed that a supplementation with a specific probiotic strain of bacteria, Lacticaseibacillus rhamnosus HA-114 (HA-114), had a neuroprotective effect in several neurodegenerative disease models. This project aimed to characterize the neurophysiological aspects of a specific nematode strain expressing the Aß1-42 fragment and to validate the neuroprotective effect of HA-114 in this nematode strain as well as in the APP/PS1 murin model of AD. Following biochemical and behavioral experiments, data showed that this strain recapitulates key traits of AD: an age-dependent alteration of neuronal health and memory impairment. It was also observed that administrating HA-114 probiotic strain could attenuate age-related phenotypes of neurodegeneration and paralysis. This transgenic strain is therefore a new tool for drug screening and investigating AD’s pathogenesis. The neuroprotective effect of HA-114 observed in this strain shows potential towards an amelioration of the patient’s quality of life. Studying the effects of HA-114 in a murin model could reveal new investigation opportunities for AD’s pathogenesis and mechanisms that could one day cure this disease.

Bakgrund: Deposition av fibrillär amyloid beta 1-42 (Aβ) i hjärnan är ett välkänt kännetecken för den neurodegenerativa sjukdomen Alzheimer’s (AD). Dessa ansamlingar påverkar pericyter, en celltyp involverad i blodkärlsfunktion och upprätthållande av blodhjärnbarriären (BBB). Pericyter uttrycker både receptorn low density lipoprotein receptor related protein 1 (LRP-1) till vilken Aβ1-42 binder, och proteoglykanet NG2. NG2 har stor betydelse för pericyters samspel med endotelceller och i sin lösliga form (sNG2) främjar den angiogenes. Tidigare studier har visat att mängden NG2 som klyvs från pericyter förändras när de stimuleras med Aβ1-42. Syfte: Att undersöka om Aβ1-42 påverkar NG2 klyvning via LRP-1 Metod: Human brain vascular pericytes (HBVP) stimulerades med monomera, oligomera och fibrillära Aβ1-42 preparationer. Uttrycket av LRP-1 tystades med small interfering (si) LRP-1 och knockdown efficiency analyserades med Western Blot (WB). Även Aβ1-42 preparationer undersöktes med WB. Cellviabilitet mättes med laktatdehydrogenas (LDH) test och proteininnehåll med Bradford analys. Slutligen mättes mängden sNG2 i pericytmedium med hjälp av enzyme-linked immunosorbant assay (ELISA) baserad på electrochemiluminescence (Mesoscale). Resultat: Preparationerna med monomer och oligomer Aβ1-42 ökade NG2 klyvning. Denna Aβ1-42 inducerade ökning försvann när cellernas LRP-1 tystats. Aβ1-42 fibrillpreparationerna inhiberade däremot NG2 klyvningen oavsett närvaro av LRP-1. Aβ1-42 monomerpreparationer inducerade celldöd hos HBVP med LRP-1 men inte hos de HBVP där LRP-1 tystats, och cellviabiliteten hos HBVP ökade hos celler som stimulerats med Aβ1-42 fibrillpreparation och där LRP-1 tystats. Konklusion: Resultaten visar att Aβ1-42 monomer och oligomer påverkar NG2 klyvning via LRP-1. Däremot verkar Aβ1-42 fibrill istället påverka NG2 klyvning via en annan signalväg. Studien belyser hur Aβ1-42 kan påverka pericyter, vilket kan föreligga vaskulära förändringar kopplade till AD patologi. / Background: The deposition of fibrillar amyloid beta 1-42 (Aβ) in the brain is a well-known characteristic for the neurodegenerative Alzheimer’s disease (AD). These accumulations affect pericytes, a cell type implicated in vessel function and maintenance of the blood-brain barrier (BBB). Pericytes express both the receptor low-density lipoprotein receptor related protein 1 (LRP-1), to which Aβ1-42 binds, and the proteoglycan NG2. NG2 is important for the interaction between pericytes and endothelial cells and in its soluble form (sNG2) it promotes angiogenesis. Earlier studies have shown that the amount of NG2 shed from pericytes is altered when stimulated with Aβ1-42. Purpose: To investigate whether the Aβ1-42 influence on NG2 shedding is mediated via LRP-1. Method: Human brain vascular pericytes (HBVP) were stimulated with monomeric, oligomeric and fibrillar preparations of Aβ1-42. Expression of LRP-1 was knocked down by small interfering (si) LRP-1 silencing and knockdown efficiency was analysed with Western blot (WB). Aβ1-42 preparations were also analysed with WB. Cell viability was measured with lactate dehydrogenase (LDH) test and protein concentrations were determined with Bradford assay. Finally the amount of sNG2 in pericytemedium was measured with enzyme-linked immunosorbant assay (ELISA) baserad på electrochemiluminescence (Mesoscale) Results: Monomer and oligomer Aβ1-42 increased NG2 shedding, this Aβ1-42 induced increase was not found in HBVP with a silenced LRP-1. In contrast, fibrillar Aβ1-42 inhibited NG2 shedding regardless of LRP-1 presence. Monomer Aβ1-42 preparations induced cell death of HBVP with LRP-1 but not of HBVP without LRP-1, and cell viability of HBVP lacking LRP-1 was increased after fibrillar Aβ1-42 exposure. Conclusion: The results indicate a monomeric and oligomeric Aβ1-42 induced impact on NG2 shedding via LRP-1. However it appears as if fibrillar Aβ1-42 doesn’t affect NG2 shedding via LRP-1 but rather inhibits the process via another unknown receptor. The study highlights how Aβ1-42 can affect pericytes, which may underlie the vascular changes linked to AD pathology.

No description available.

Introduction: Alzheimer's disease is a neurodegenerative disease that initially manifests itself primarily as impaired short-term memory and impaired language ability. The course of the disease is mainly due to an atrophy in the brain that can be attributed to the protein amyloid B and tau. Monoclonal antibodies that target Alzheimer's disease often have a high rate of cerebral edema, where proteinaceous fluid leaks into the extracellular space of the brain and creates edema. Some of the most common symptoms for amyloid-related imaging abnormalities (ARIA-E) are headache, dizziness, and blurred vision. In a few cases, patients with ARIA-E need to be hospitalized for observation, but most show a decline in ARIA-E within one to two months. Objective: To investigate the frequency of ARIA-E in clinical studies of monoclonal antibodies to patients with Alzhiemer's disease and to investigate the role of the ApoE4 allele in the development of ARIA-E. Method: Literature review of five RCT studies based on four different monoclonal antibodies. PubMed was used to search for the RCT-studies. Results: ARIA-E varies between different types of antibodies. ARIA-E usually occurs early in treatment when the degree of amyloid b is highest in the brain. Most cases are asymptomatic and treatment resumes within 1-2 months. Conclusion: Aria-E frequency correlates strongly with dose strength as well as APOE4 -status and most of the incidences are asymptomatic. With the right titration and individually selected drugs as well as individual dosages a safe care can be established for patients with Alzheimer's disease. If treatment is initiated at an early stage, the risk of side effects is reduced and more neurons can be saved from atrophy. The combination of several different types of medicine will further reduce the risk of ARIA-E.

No description available.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1-42) and pGlu-Aβ(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1-42) and pGlu-Aβ(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Dans la maladie d'Alzheimer (MA), les altérations du sommeil sont parmi les premiers symptômes cliniques observés. Il existe une littérature croissante indiquant que les oligomères amyloïde-bêta (Aβo) sont particulièrement neurotoxiques et leur contribution spécifique aux troubles du sommeil reste à définir. Récemment, une dérégulation du métabolisme lipidique a été signalée dans le cerveau des patients atteints de la MA et des modèles animaux. De plus, l'inhibition de la stéaroyl-CoA désaturase (SCD), une enzyme de conversion lipidique, a montré qu'elle rétablissait la mémoire chez les souris triple transgénique (3xTg), modèle de la MA. Dans le cerveau, les astrocytes régulent la synthèse de lipides et des altérations des astrocytes et de leurs fonctions ont été signalées chez les patients atteints de la MA et les modèles animaux. De plus, les astrocytes ont été impliqués dans la régulation du sommeil. Cependant, la relation entre les troubles du sommeil, les astrocytes et le métabolisme lipidique reste à explorer dans la MA. Par conséquent, cette thèse avait deux objectifs : définir l'effet d'Aβo sur différentes variables de sommeil et vérifier si la SCD contribue aux troubles du sommeil chez les 3xTg et si cela s'associe à des modifications de la fonction astrocytaire. Pour cela, des injections chroniques (6 jours) d'Aβo dans l'hippocampe ont été réalisées chez des rats mâles de type sauvage (WT pour « Wild-type ») et des mesures électroencéphalographiques (EEG) ont été effectuées pour définir les altérations veille/sommeil. De plus, des souris femelles WT et 3xTg ont reçu un traitement intracérébroventriculaire de 28 jours avec un inhibiteur de la SCD (SCDi) ou un véhicule, et des enregistrements EEG ont été réalisés après les 28 jours de traitement. Des sections de cerveau ont été soumises à une coloration pour les marqueurs astrocytaires, tels que la protéine acide fibrillaire gliale (GFAP) et l’aldéhyde déshydrogénase 1 de type L1 (ALDH1L1), en vue de réaliser une quantification cellulaire et/ou une évaluation morphologique dans l'hippocampe, l'hypothalamus latéral et le thalamus. Les résultats montrent que le temps passé en éveil, en sommeil lent (SL) et en sommeil paradoxal (SP) a été préservé chez les rats injectés avec l'Aβo. Cependant, l'activité spectrale de l'EEG pendant l'éveil a été augmentée par l’Aβo pour l'activité des ondes lentes (0,5-5 Hz) et l'activité bêta de basse fréquence (16-20 Hz), tandis qu'elle a été diminuée pendant le SL pour l'activité thêta (5-9 Hz) et alpha (9-12 Hz). De plus, le ratio thêta/activité à ondes lentes a été diminué pendant l'éveil et le SL. De plus, six jours d’injection d’Aβo ont été nécessaires pour observer les changements spectraux chez le rat. Concernant le second objectif, le traitement SCDi n’a pas restauré la diminution du temps d'éveil et l'augmentation du temps passé en SL chez les souris 3xTg. L'activité EEG rythmique et arythmique a été considérablement altérée chez les souris 3xTg pour tous les états d'éveil/sommeil, et le SCDi a modifié significativement ces phénotypes de manière différente chez les souris mutantes par rapport aux souris WT. Les densités de cellules positives pour GFAP et ALDH1L1 ont été augmentés dans l'hippocampe et l'hypothalamus/thalamus, respectivement, et le SCDi a atténué l'augmentation dans la région CA1 en particulier. Les résultats confirment la présence d'altérations du sommeil dans les modèles de la MA et suggèrent que les phénotypes de sommeil pourraient servir de marqueur non invasif précoce de la MA. Cependant, malgré le ciblage du métabolisme lipidique par l'inhibition de la SCD, les diverses perturbations du cycle éveil/sommeil chez les souris 3xTg n’ont pas été significativement rétablies. Mais il semble que ce traitement soit capable de renverser les modifications astrocytaires observées dans l'hippocampe. Ce travail contribuera à la compréhension de la physiopathologie liée à la MA et aux troubles du sommeil associés. / In Alzheimer's disease (AD), sleep disturbances are among the earliest clinical symptoms observed. There is a growing body of evidence indicating that soluble amyloid-bêta oligomers (Aβo) are particularly neurotoxic, and their specific contribution to sleep disorders is yet to be defined. Recently, dysregulation of lipid metabolism has been reported in the brains of AD patient and animal models. Furthermore, inhibition of stearoyl-CoA desaturase (SCD), a lipid-converting enzyme, has shown to restore memory in triple transgenic (3xTg) mice, an AD model. In the brain, astrocytes regulate lipid synthesis, and alterations in astrocytes and their function have been reported in AD patient and animal models. Additionally, astrocytes have been implicated in the regulation of sleep. However, the relationship between sleep disturbances, astrocytes, and lipid metabolism remains to be explored in AD. Therefore, this thesis had two objectives: to define the effect of Aβo on various sleep variables and to investigate whether SCD contributes to sleep disturbances in 3xTg mice and if this is associated with changes in astrocytic function. For this purpose, chronic injections (6 days) of Aβo into the hippocampus were performed in male wild-type (WT) rats, and electroencephalographic (EEG) measurements were conducted to assess wake/sleep alterations. Additionally, female WT and 3xTg mice received a 28-day intracerebroventricular treatment with an SCD inhibitor (SCDi) or a control, and EEG recordings were made after the 28-day treatment. Brain sections were stained for astrocytic markers, such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1), to perform cell counting and/or morphological evaluation in the hippocampus, lateral hypothalamus, and thalamus. The results show that the time spent awake, in slow-wave sleep (SWS), and in rapid eye movement (REM) sleep was preserved in rats injected with Aβo. However, spectral EEG activity during wakefulness was increased by Aβo for slow-wave (0.5-5 Hz) and low-frequency beta (16-20 Hz) activity, while it was decreased during SWS for theta (5-9 Hz) and alpha (9-12 Hz) activity. Additionally, the theta/SWA ratio was decreased during wakefulness and SWS. Furthermore, six days of Aβo injection were required to observe spectral changes in rats. Regarding the second objective, SCDi treatment did not restore the reduction in wake time and the increase in SWS time in 3xTg mice. Rhythmic and arrhythmic EEG activity was significantly altered in 3xTg mice for all wake/sleep states, and SCDi significantly modified these phenotypes differently in mutant mice compared to WT mice. GFAP- and ALDH1L1-positive cell densities were increased in the hippocampus and lateral hypothalamus/thalamus, respectively, and SCDi attenuated the increase in the CA1 region only. The results confirm the presence of sleep alterations in Alzheimer's disease models and suggest that sleep phenotypes could serve as a non-invasive marker of early Alzheimer's disease. However, despite targeting lipid metabolism through SCD inhibition, the various disturbances in the wake/sleep in 3xTg mice were not significantly restored. Nevertheless, it appears that this treatment can reverse astrocytic changes observed in the hippocampus. This work will contribute to the understanding of the pathophysiology related to Alzheimer's disease and associated sleep disturbances.

No / Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

There are two kinds of protein aggregates associated with Alzheimer’s disease: amyloid-beta and tau aggregates. Protein ligands are molecules with the ability to bind to these pathologicalprotein accumulations, and if the ligands are fluorescent, they can be used to detect the aggregates they’re bound to. The ligands can be selective and only bind to one kind of protein aggregate, or they can be general and bind to both kinds. PI-2620 is a ligand selective for tau aggregates, and in this thesis, three analogues of PI-2620 were synthesized and determined to be functional fluorescent protein ligands. Two of them, designated A1 and B1, were selective for amyloid-beta aggregates, while the third ligand, A2, obtained by ester-hydrolysis of A1, would bind to both tau and amyloid-beta aggregates. This finding suggests that introducing a charge to a ligand lowers its selectivity, since the staining experiments were carried out in a buffered solution at pH 7.4, where ligand A2 might be partially charged.