With 9.9 million new dementia cases each year, Alzheimer’s and Parkinson’s disease (AD and PD) are the most prevalent form of neurodegenerative disorder (NDG) affecting the aging population. Despite years of pharmaceutical research, no cure is yet available. Most neuropathological aspects of these diseases are extremely complex but the study of the rare genetic cases allowed to model these diseases in animals and uncover key pathophysiological processes. Transgenic Drosophila NDG models have been used for in vivo studies for many years with a range of relevant phenotypes. The cellular and molecular biology of the Central Nervous System, as well as the mechanisms underlying neurodegeneration, are well conserved between Drosophila and Humans (with a 75% of human disease-related genes having homologs in flies). Most NDG studies are performed in the aging flies. However, there are reports of measurable phenotypes for a variety of AD and PD models in juvenile Drosophila melanogaster (larval stage) with an unexploited considerable potential for drug discovery and screening for this outstanding model. Here I sought to develop a new assay for research into NDGs that focus on the earliest phenotypes. During this Ph.D. project a customized crawling assay apparatus was developed, for the assessment of locomotor ability in humanised larval Drosophila (overexpressing human proteins/peptides linked to AD and PD). A locomotor phenotype was identified in larvae overexpressing different variation of Amyloid-β42, tau and α-Synuclein pan neutrally: these animals crawl on agarose surface at a reduced mean speed when compared to controls. The defect was proven partially rescuable by administration of Tacrine and Methylene Blue, renewing the importance of such models for future applications in drug discovery and screening. The motor impairment supports the hypothesis of a neurotoxic effect of the protein/peptide. Thus, to test this further, the overexpression of the human transgenes was restricted to neurons involved in larval olfaction (olfactory impairment is often the earliest symptom in PD and AD) and odour associated learning tasks (both PD and AD are characterized by severe cognitive dysfunction). Interestingly, larvae overexpressing the Amyloid-β42 ARC peptide in the Olfactory Sensory Neurons showed a subtle navigation defect during chemotaxis (in 1-Hexanol odour gradient) that could possibly be addressed to premature neural habituation to the olfactory stimulus. Furthermore, the overexpression of the peptide in the larval Mushroom Bodies influenced the performances of the animals in associative learning tasks. Lastly, using immunohistochemistry and confocal imaging techniques I showed that the gross morphology of neurons is not altered by the targeted overexpression of the Amyloid-β42 ARC. Even though physiological studies are required to characterize the chemosensory/learning defect shown by the Amyloid-β42 ARC larvae, this Ph.D. work further confirms that the effects of the overexpression of the human transgenes are robust and measurable already at larval stage. These findings may also be relevant to the development of new, fast, and cost-effective compound screening procedures, for applications in early stages of the drug discovery process.

Alzheimer’s disease (AD) is the most common form of dementia and a growing disease burden that has seen pharmacological interventions primarily fail. Instead, it has been suggested that preventative measures such as a healthy diet may be the best way in preventing AD. Prebiotics are one such potential measure and are fermented into metabolites by the gut microbiota and acting as gut-brain axis components, beneficially impact the brain. However, the impact of prebiotics in AD prevention is unknown. Here we show that the prebiotic inulin increased multiple gut-brain axis components such as scyllo-inositol and short chain fatty acids in the gut, periphery, and in the case of scyllo-inositol, the brain. We found in E3FAD and E4FAD mice fed either a prebiotic or control diet for 4-months, that the consumption of the prebiotic inulin can beneficially alter the gut microbiota, modulate metabolic function, and dramatically increase scyllo-inositol in the brain. This suggests that the consumption of prebiotics can beneficially impact the brain by enhancing metabolism, helping to decrease AD risk factors.

Age-related neurologic disease is a significant and growing burden on our society. Although the largest share of research effort has typically been devoted to the common neurodegenerative illnesses (such as Alzheimer’s disease, or AD), the reality is that nearly all cases of neurodegenerative disease possess elements of mixed pathology. Vascular contributions to cognitive impairment and dementia (VCID) is a complex form of dementia, combining aspects of vascular disease and other forms of dementia, such as Alzheimer’s disease. This pathology is heterogeneous and can include cerebral amyloid angiopathy (CAA), hemorrhages, white matter infarcts, and changes to the neurovascular unit. Given the heterogeneous nature of VCID, we hypothesized that we could further elucidate mechanisms that drive dementia in VCID by examining pathology in mouse models and use this data to guide the study of human autopsy cases. Using a mouse model of VCID, we identified NHE1, a sodium hydrogen exchanger that was upregulated in these mice, as a possible candidate for a factor involved in cerebrovascular disease in humans. We saw a significant age effect of NHE1 in cases with Down syndrome (DS), leading us to further examine cerebrovascular pathology in individuals with DS. People with DS are at a high risk of developing cognitive impairment and dementia after the age of 50. In fact, virtually all adults with DS develop the neuropathology for an AD (beta-amyloid (Aß) senile plaques and tau neurofibrillary tangles) diagnosis by the age of 40 due to a triplication of chromosome 21. We found that these individuals develop CAA and microhemorrhages as a function of age, and that these rates are as severe as sporadic AD, despite an age difference of ~30 years. We also found that individuals with DS have different microglial morphologies than controls or individuals with AD. This data indicates that people with DS develop significant cerebrovascular and AD pathology, indicative of VCID. Overall, we found that mixed pathologies, specifically VCID, is an important contributor to the development of dementia and should be studied further to better understand how this pathology drives cognitive impairment. Further, it is clear that mouse models map imperfectly onto complex human diseases, and that significant work remains to be done towards achieving an adequate model of VCID.

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.

Notre société occidentale fait face actuellement à une hausse de l’espérance de vie, ce qui amène à un vieillissement de la population. Malgré les avancées médicales, il s’opère une dégradation des fonctions cognitives et des capacités physiques, responsable d’une baisse de la qualité de vie et de la perte d’autonomie. De plus, le nombre de pathologies dégénératives, dont le premier facteur de risque est l’âge, ne cesse d’augmenter. A l’heure actuelle, il n’existe encore aucun moyen pharmacologique permettant de contrer efficacement le déclin cognitif et physique lié à l’âge. Ainsi, l’objectif de ce travail de thèse était de tester l’impact de méthodes non-pharmacologiques sur la cognition, particulièrement au niveau de la mémoire de travail et des fonctions exécutives, la qualité de vie, et l’autonomie. Nous nous sommes concentrés sur les entraînements cognitifs et physiques, en émettant l’hypothèse principale qu’une combinaison de ces deux sphères au sein d’un même entraînement serait plus efficace qu’un entraînement seul (i.e. cognitif ou physique) dans la mesure où ils engendreraient des bénéfices différents mais complémentaires. Ce travail de thèse s’organise donc autour de trois études : l’Etude 1, qui a pour but d’investiguer l’impact d’un entraînement cognitif simple comparé à un entraînement combiné cognitif-et-physique sur la cognition ; l’Etude 2, qui compare l’impact de ces deux mêmes entraînements mais cette fois à un niveau fonctionnel cérébral ; l’Etude 3 qui investigue l’impact d’une prise en charge multidimensionnelle dans la maladie d’Alzheimer. Les résultats de ce travail de thèse nous montrent, en ce qui concerne le vieillissement normal, des bénéfices des deux types d’entraînement au niveau comportemental, en termes de transfert et de maintien à long-terme. En revanche, si l’entraînement combiné a engendré un transfert plus important, l’entraînement cognitif seul produit un meilleur maintien des bénéfices dans le temps. Par rapport au niveau fonctionnel cérébral, des bénéfices ont été montrés sur la composante P100, qui reflète le recrutement attentionnel uniquement pour le groupe d’entraînement cognitif seul. Ensuite, nous avons observé des bénéfices dans le vieillissement pathologique au niveau de la cognition. Pour conclure, ce travail de thèse nous a permis de montrer des effets bénéfiques des entraînements cognitifs et/ou physiques dans le vieillissement normal et pathologique, même si à l’heure actuelle nous ne pouvons pas conclure par une supériorité de l’entraînement combiné comparé à un entraînement cognitif. / Our western society is currently facing an increase in life expectancy, which is leading to a growing aging population. Despite medical advances, there is a deterioration of cognitive functions and physical abilities, which is responsible for a decline in life quality and a loss of autonomy. In addition, the number of neurodegenerative diseases, whose first risk factor is age, is increasing. Today, there is no pharmacological means to effectively counter agerelated cognitive and physical decline. Thus, the objective of this thesis was to test the impact of non-pharmacological methods on cognition, particularly on working memory and executive functions, quality of life, and autonomy. We focused on cognitive and physical training, with the main hypothesis that a combination of these two aspects within the same training would be more effective than a single workout (ie cognitive or physical) insofar as they would generate different but complementary benefits. This thesis work is organized around three studies : Study 1, which aims to investigate the impact of a simple cognitive training compared to a combined cognitive-and-physical training on cognition ; Study 2, which compares the impact of these two same trainings but this time at a cerebral functional level ; Study 3 which investigates the impact of multidimensional intervention in Alzheimer's disease. The results of this thesis work showed, with regard to normal aging, the benefits of both types of training at the behavioral level, in terms of transfer and long-term maintenance. On the other hand, if combined training has resulted in greater transfer, cognitive training alone produces better benefits maintenance over time. Relative to brain function level, benefits were shown on the P100 component, which reflects attentional recruitment solely for the cognitive training group alone. Then, we observed benefits in pathological aging at the level of cognition. To conclude, this thesis work allowed us to show the beneficial effects of cognitive and / or physical training in normal and pathological aging, even if at present we cannot conclude for the superiority of combined training compared to cognitive training.

Small fluorescent probes are important tools when studying protein aggregates involved in different neurodegenerative diseases, such as Alzheimer’s disease. Luminescent conjugated oligothiophenes have been developed and shown to be excellent ligands when studying morphology among amyloids, due to their conjugated thiophene backbone that provides them with unique photophysical properties. This kind of probes are being developed successively to enhance the specificity of their biological targets. In this project, luminescent conjugated oligothiophenes functionalized with amino acids, so called proteophenes, have been synthesized to investigate their optical properties. Since amino acids are chiral molecules, the possibility of induced chirality to the thiophene backbone was examined, as well as the proteophenes ability to work as amyloidospecific ligands for the study of protein aggregates. The synthesis of four different proteophenes are presented in this report, along with analysis results of their photophysical properties.

<p>Alzheimer Disease (AD) is the most common neurodegenerative disorder in the world. One of the neuropathological hallmarks of AD is the senile plaques in the brain. The plaques are mainly composed of the amyloid β (Aβ) peptide. Aβ is generated from the amyloid precursor protein, APP, when it is first cleaved by the β-secretase and subsequently the γ-secretase complex. The γ-secretase complex cleaves at different sites, called γ and ε, where the γ-cleavage site generates Aβ peptides of different lengths and ε-cleavage generates the APP intracellular domain (AICD). The two major forms of Aβ is 40 and 42 amino acids long peptides, where the latter is more prone to aggregate and is the main component in senile plaques. The γ-secretase complex is composed of four proteins; Pen-2, Aph-1, nicastrin and presenilin (PS). The PS protein harbours the catalytic site of the complex, where two aspartate residues in position 257 and 385 (Presenilin 1 numbering) are situated. Most Familial AD (FAD) mutations in the PS gene cause a change in the γ-cleavage site, leading to a shift from producing Aβ40 to the longer more toxic variant Aβ42. Frequently, this often leads to impairments of the AICD production. Another substrate for the γ-secretase complex is Notch. It is important to maintain the Notch signaling since an intracellular domain (NICD) is formed after cleavage by the γ-secretase complex in the membrane (S3-site) and this domain is involved in transcription of genes important for cell fate decisions.</p><p>It has been reported that certain APP luminal juxtamembrane mutations could drastically alter Aβ secretion, however their effect on AICD production remains unknown. In this study we want to analyse wether the juxtamembrane region is important for the AICD production. To gain more insight into the luminal juxtamembrane function for γ-secretase-dependent proteolysis, we have made a juxtamembrane chimeric construct. A four-residue sequence preceding the transmembrane domain (TMD) of APP (GSNK), was replaced by its topological counterpart from the human Notch1 receptor (PPAQ). The resulting chimeric vector C99GVP-PPAQ and the wildtype counterpart were expressed in cells lacking PS1 and PS2 (BD8) together with PS1wt. We observed that the chimeric construct did not alter production of AICD when using a cell based luciferase reporter gene assay monitoring AICD production. We also introduced a PS1 variant lacking a big portion of the large hydrophilic loop, PS1∆exon10, since our group has previously observed that this region affect Aβ production¹⁴³. We found that the absence of the large hydrophilic loop in PS1 gave a 2-fold decrease in AICD-GVP formation from C99GVPwt compared to PS1wt.  The activity of PS1wt and PS1Δexon10 using C99GVP-PPAQ as a substrate gave similar result as the C99GVPwt substrate, i.e. a 2-fold decrease in AICD-GVP formation when comparing PS1Δexon10 with PS1wt. From this data we therefore suggest that the four residues in the juxtramembrane domain (JMD) (GSNK) is not altering ε-cleavage of APP when changed to Notch1 counterpart, PPAQ. Furthermore, we also show that the 2-fold decrease in AICD-production by the PS1Δexon10 molecule is not changed between the two substrates C99GVPwt and C99GVP-PPAQ. This indicates that the luminal region of APP is not directly involved in the ε-site processing. If the luminal region is affecting processing in the γ-cleavage sites, remains however to be investigated.</p>

The Arctic mutation in the Amyloid-β (Aβ) domain of the Amyloid-β precursor protein (APP) causes Alzheimer’s disease (AD) and confers unique biochemical characteristics to Aβ peptides. The aims of this thesis were to evaluate a transgenic model with the Arctic mutation, and to use it to gain new insights into the mechanisms of early (pre-plaque) and late-stage Aβ pathogenesis in AD. The Arctic mutation made Aβ more prone to aggregate, to accumulate in intracellular compartments and to form extracellular plaques when the models tg-ArcSwe and tg-Swe were compared. By inhibiting APP processing genetically or pharmacologically, the intraneuronal granular immunoreactivity with antibodies binding the Aβ domain was shown to largely represent Aβ, and not APP or APP-fragments. At two months of age, the intracellularly accumulated Aβ decreased rapidly, likely because it was still accessible to intracellular clearance. Extracellular Aβ deposits emerged at 5-6 months of age and the amyloid fibril structure was more compact than in tg-Swe. Moreover, Aβ deposits in tg-ArcSwe were more resistant to chemical extraction than those of established models carrying the Swedish APP mutation only, e.g. tg-Swe mice. The stability of deposits better reflects the biochemistry of senile plaques in AD. Thus, the tg-ArcSwe model may better predict the outcome of clinical trials, particularly therapies designed to enhance clearance of Aβ aggregates and deposits. Postmortem brain of Arctic mutation carriers contained extensive parenchymal plaque pathology. Differential immunostaining patterns with C- and N-terminal Aβ antibodies revealed a subset of plaques that were unique to the brains of Arctic mutation carriers. Aβ deposits in the cerebral vessel walls were congophilic and mainly composed of full-length Aβ. In contrast, N-terminally truncated Aβ was more prominent in the parenchymal plaques, all of which essentially lacked amyloid cores. A heterogeneous assembly of mutant and wild-type Aβ was shown to favor the formation of diffuse deposits in bitransgenic mice, and such mechanisms may at least partly explain observations of plaques lacking amyloid cores in postmortem Arctic mutant brain. In the bitransgenic mice, a low level of Arctic Aβ was sufficient to facilitate aggregation of wild-type Aβ. This observation, but also our findings of differences in amyloid fibril structure in tg-ArcSwe and tg-Swe, further highlights similarities between AD and prion disorders in which PrPsc refolds PrPc and facilitates fibril formation. / (Faculty of medicine)

Bakgrund: Idag lever minst 150 000 personer med demens i Sverige, år 2050 beräknas antalet ha fyrdubblats. Insatserna för personer med demens ska ge dem den hjälp de behöver i det sociala samspelet och ge dem en känsla av trygghet. Att som människa bli sedd och bekräftad ökar chansen för god hälsa, och eftersom hunden är expert på att tolka kroppsspråk och subtila signaler som människor lätt missar erbjuder den ett speciellt sällskap. Syfte: Beskriva hundens roll inom demenssjukvård. Frågeställningar: Hur kan hunden användas i omvårdnaden av personer med demenssjukdom? Vilka effekter har interventioner med hund på personer med demenssjukdom? Metod: Systematisk litteraturstudie. Resultat: Det finns i huvudsak två olika sätt att använda intervention med hund inom demenssjukvård, Animal Assisted Activity (AAA) och Animal Assisted Therapy (AAT). AAA fokuserar på trivsel och social samvaro medan AAT har fokus på individuell behandling. Både psykologiska och fysiologiska effekter hos personer med demens kunde påvisas av interventioner med hund. Effekter på socialt beteende, beteendeproblem, kognitiva förmågor, upplevelse av det dagliga livet samt effekter på rörelse och stress kunde uppvisas. Inga effekter på läkemedelsanvändning observerades. Slutsats: Studieresultatet visar på att det är syftet med hundinterventionen som bestämmer om det är AAA eller AAT som bör användas. Vidare tyder resultatet på att det är troligt att socialt beteende och fysisk rörelse ökar av hundinterventioner, samt att beteendeproblem och stress kan minska. Studieresultatet antyder inte någon effekt på läkemedelsanvändningen.

The disturbances and abnormalities occurring in the components of the Acetylcholine (ACh) neurocycle are considered one of the main features of cholinergic sicknesses like Parkinson’s and Alzheimer’s diseases. A fundamental understanding of the ACh neurocycle is therefore very critical in order to design drugs that keep the ACh concentrations in the normal physiological range. In this dissertation, a novel two-enzyme-two-compartment model is proposed in order to explore the bifurcation, dynamics, and chaotic characteristics of the ACh neurocycle. The model takes into consideration the physiological events of the choline uptake into the presynaptic neuron and the ACh release in the postsynaptic neuron. In order to approach more realistic behavior, two complete kinetic mechanisms for enzymatic processes pH-dependent are built: the first mechanism is for the hydrolysis reaction catalyzed by the acetylcholinesterase (AChE) and the other is for the synthesis reaction catalyzed by the cholineacetyltransferase (ChAT). The effects of hydrogen ion feed concentrations, AChE activity, ChAT activity, feed ACh concentrations, feed choline concentrations, and feed acetate concentrations as bifurcation parameters, on the system performance are studied. It was found that hydrogen ions play an important role, where they create potential differences through the plasma membranes. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the activity of AChE through a certain range of their concentrations, where the activity of AChE is inhibited completely after reaching certain values. A detailed bifurcation analysis over a wide range of parameters is carried out in order to uncover some important features of the system, such as hysteresis, multiplicity, Hopf bifurcation, period doubling, chaotic characteristics, and other complex dynamics. The effects of the feed choline concentrations and the feed acetate concentrations as bifurcation parameters are studied in this dissertation. It is found that the feed choline concentrations play an important role and have a direct effect on the ACh neurocycle through a certain important range of the parameters. However, the feed acetate concentrations have less effect. It is concluded from the results that the feed choline is a more important factor than the feed acetate in ACh processes. The effects of ChAT activity and the choline recycle ratio as bifurcation parameters, on the system performance are investigated. It was found that as the ChAT activity increases, ACh concentrations in compartments 1 and 2 increase continuously. The effect of the choline recycle ratio shows that choline reuptake plays a very critical role in the synthesis of ACh in compartment 1, where it supplies the choline as a substrate for the synthesis reaction by ChAT. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the choline recycle ratio through a certain range of the choline recycle ratio; then, they become constant as the choline recycle ratio increases further. It is concluded from our results that choline uptake is the rate limiting step in the ACh processes in both compartments in comparison to ChAT activity. Based on partial dissociation of the acetic acid in compartments 1, and 2 of the ACh cholinergic system, the two-parameter continuation technique has been applied to investigate the pH range to be closer to physiological ranges of pH values. In addition, static/dynamic solutions of the ACh cholinergic neurocycle system based on feed choline concentration as the main bifurcation parameter in both compartments have been investigated. The findings of the above studies are related to the real phenomena occurring in the neurons, like periodic stimulation of neural cells and non-regular functioning of ACh receptors. It was found that ACh, choline, acetate, and pH exist inside the physiological range associated with taking into consideration the partial dissociation of the acetic acid. The disturbances and irregularities (chaotic attractors) occurring in the ACh cholinergic system may be good indications of cholinergic diseases such as Alzheimer’s and Parkinson’s diseases. The results have been compared to the results of physiological experiments and other published models. As there is strong evidence that cholinergic brain diseases like Alzheimer’s disease and Parkinson’s disease are related to the concentration of ACh, the present findings are useful for uncovering some of the characteristics of these diseases and encouraging more physiological research.