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In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques. In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap. In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized. / QC 20101112

The aim of the present thesis was twofold, first to map the semantic memory decline in Alzheimer patients over time, second to take the patient’s perspective and create a multifaceted picture of the individual with Alzheimer’s disease through the study of memory, awareness, central coherence and emotions. Further issues concerned how Alzheimer individuals handled their cognitive erosion in everyday life and if they were well calibrated with their spouse in disease related matters. Two studies were performed, the first involved a longitudinal study of sematic deterioration, the second had a mixed methods design involving both quantitative and qualitative measures as in neuropsychological assessment and interviews. Through the longitudinal study it could be concluded that the nature of semantic deterioration is best described as loss of memory information rather than problems in accessing the information. It was further concluded that semantic concepts gradually lose their specific features during course of illness. The results from the second study revealed that the Alzheimer individuals were aware of their disease although they could not foresee the implications of their cognitive shortcomings in their everyday life. They evidenced weak central coherence, in that they were unable to infer details into a meaningful whole. This implies that they perceive their surrounding world in a fragmented way as consisting of separate objects rather than a comprehensible context. Concerning emotions it was found that they responded to negatively valenced words in the same way as normal ageing individuals, although being impaired in their response to positively and neutral words. Finally, the Alzheimer individuals and their spouses were not well calibrated regarding disease related issues. The findings of the present thesis have important clinical implications and gives valuable input to the understanding of the individual with Alzheimer’s disease. / Föreliggande avhandling hade två huvudsyften; dels att kartlägga försämringen av semantiskt minne hos Alzheimerpatienter över tid; dels att ta patientens perspektiv och skapa en mångfacetterad bild av individen med Alzheimers sjukdom. Fyra begrepp studerades i relation till detta, nämligen minne, medvetande, central koherens och emotioner. Ytterligare aspekter som studerades var hur Alzheimerindivider hanterar sina kognitiva tillkortakommanden i sin vardag samt hur väl kalibrerade de är med sin make/maka angående sjukdomsrelaterade frågor. Två studier genomfördes varav den första var en longitudinell studie av semantisk minnesförsämring och den andra hade en ’mixed methods’ design, inkluderande både kvantitativa (neuropsykologiska tester) och kvalitativa (intervjuer) metoder. Resultaten från den longitudinella studien avslöjade att semantiska begrepp som inte längre är välrepresenterade hos individen har gått förlorade snarare än att det skulle vara från om att individerna inte kan plocka fram informationen. Vidare kunde konstateras att semantiska begrepp gradvis tappar sina nyanser i takt med att sjukdomen fortskrider. Den andra studien visade att Alzheimerindividerna var medvetna om sin sjukdom och sina minnesproblem men att de inte kunde förutsäga vilka problem deras kognitiva tillkortakommanden skapade i deras vardag. De konstaterades ha svag central koherens vilket innebär att de ser sin omgivning på ett fragmentariskt sätt utan att kunna få ihop de olika objekten runt omkring sig till en meningsfull kontext. Vad gäller emotioner så kunde konstateras att Alzheimerindivider reagerar likadant som normalt åldrande individer på negativt laddade ord men att de är signifikant försämrade i förmågan att känna igen positiva och neutrala ord. Alzheimerindividerna och deras make/maka var inte väl kalibrerade vad gäller sjukdomsrelaterade frågor. Avhandlingens resultat har viktiga kliniska implikationer och ger ett värdefullt bidrag till förståelsen av en individ med Alzheimers sjukdom.

Η νόσος Αλτσχάιμερ καταλαμβάνει την πρωτοκαθεδρία στις μη αναστρέψιμες άνοιες με τους επιδημιολογικούς της δείκτες να αυξάνονται όσο μεγαλώνει το προσδόκιμο ζωής του ανθρώπου. Η χρήση φαρμάκων, με αρχική στόχευση άλλη πάθηση, στη νόσο Αλτσχάιμερ αποκαλείται φαρμακευτικός επαναπροσδιορισμός και προσφέρει σαφή πλεονεκτήματα στην ασφάλεια, την ταχύτητα και το κόστος ανάπτυξης μίας εν δυνάμει θεραπείας, ιδιαίτερα όταν η μέχρι σήμερα αντιμετώπιση της πάθησης περιορίζεται στην καθυστέρηση εξέλιξης της βλάβης και τις δευτερογενείς εκδηλώσεις. Στην παρούσα εργασία, εκμεταλλευτήκαμε εργαλεία φαρμακευτικής επαναστόχευσης που βασίζονται στις γονιδιακές υπογραφές πέντε μελετών μικροσυστοιχιών της νόσου Αλτσχάιμερ. Καίριο στάδιο στη συγκρότηση γονιδιακών υπογραφών είναι ο προσδιορισμός της διαφορικής έκφρασης των γονιδίων. Εφαρμόζοντας τρεις διαφορετικές τεχνικές (Limma, ChDir, mAP-KL) για το σκοπό αυτό και τοποθετώντας τα αποτέλεσματα σε τέσσερα ξεχωριστά εργαλεία φαρμακευτικής επαναστόχευσης (cMap, SPIEDw, sscMap, LINCS-L1000), αναδείξαμε φάρμακα που συστηματικά αντιστρατεύονται τη νόσο. Η περαιτέρω ανάλυση σε επίπεδο χημικής δομής, λειτουργικών μονοπατιών και δικτυακής θεώρησης προσδιόρισε το μηχανισμό δράσης των φαρμάκων και πρότεινε νέα βιοδραστικά μόρια ως δυνατικές θεραπευτικές επιλογές. / Alzheimer’s disease dominates dementias of irreversible cause with alarming epidemiologic characteristics due to rise of human life expectancy. The use of initially otherwise purposed drugs in Alzheimer is described as drug repositioning and offers clear advantages in terms of safety, speed and cost issues in the development of a potential therapy, particularly when current treatments are limitited to symptoms’ delay and secondary comorbidities. In this study, we exploited drug repurposing tools based on gene signatures from five microarray experiments of Alzheimer’s disease. A fundamental step in constructing gene signatures is to define differential gene expression. For this purpose, we used three different methods (Limma, ChDir, mAP-KL) which we analyzed with four distinct drug repurposing tools (cMap, SPIEDw, sscMap, LINCS-L1000) and found drugs that systematically reverse the disease signature. Further processing of the results with regard to chemical structure, pathway and network analysis revealed the mode of the drugs’ actions and highlighted them as potential therapeutic choices for Alzheimer’s disease.

The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology. This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease. A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD. Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity. LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion. In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.

Introduction: La démence peut être causée par la maladie d’Alzheimer (MA), la maladie cérébrovasculaire (MCEREV), ou une combinaison des deux. Lorsque la maladie cérébrovasculaire est associée à la démence, les chances de survie sont considérées réduites. Il reste à démontrer si le traitement avec des inhibiteurs de la cholinestérase (ChEIs), qui améliore les symptômes cognitifs et la fonction globale chez les patients atteints de la MA, agit aussi sur les formes vasculaires de démence. Objectifs: La présente étude a été conçue pour déterminer si la coexistence d’une MCEREV était associée avec les chances de survie ou la durée de la période jusqu’au placement en hebergement chez les patients atteints de la MA et traités avec des ChEIs. Des études montrant de moins bons résultats chez les patients souffrant de MCEREV que chez ceux n’en souffrant pas pourrait militer contre l’utilisation des ChEIs chez les patients atteints à la fois de la MA et la MCEREV. L'objectif d'une seconde analyse était d'évaluer pour la première fois chez les patients atteints de la MA l'impact potentiel du biais de « temps-immortel » (et de suivi) sur ces résultats (mort ou placement en hebergement). Méthodes: Une étude de cohorte rétrospective a été conduite en utilisant les bases de données de la Régie de l’Assurance Maladie du Québec (RAMQ) pour examiner la durée de la période jusqu’au placement en hebergement ou jusqu’au v décès des patients atteints de la MA, âgés de 66 ans et plus, avec ou sans MCEREV, et traités avec des ChEIs entre le 1er Juillet 2000 et le 30 Juin 2003. Puisque les ChEIs sont uniquement indiquées pour la MA au Canada, chaque prescription de ChEIs a été considérée comme un diagnostic de la MA. La MCEREV concomitante a été identifié sur la base d'un diagnostic à vie d’un accident vasculaire cérébral (AVC) ou d’une endartériectomie, ou d’un diagnostic d'un accident ischémique transitoire au cours des six mois précédant la date d’entrée. Des analyses séparées ont été conduites pour les patients utilisant les ChEIs de façon persistante et pour ceux ayant interrompu la thérapie. Sept modèles de régression à risque proportionnel de Cox qui ont varié par rapport à la définition de la date d’entrée (début du suivi) et à la durée du suivi ont été utilisés pour évaluer l'impact du biais de temps-immortel. Résultats: 4,428 patients ont répondu aux critères d’inclusion pour la MA avec MCEREV; le groupe de patients souffrant seulement de la MA comptait 13,512 individus. Pour le critère d’évaluation composite considérant la durée de la période jusqu’au placement en hebergement ou jusqu’au décès, les taux de survie à 1,000 jours étaient plus faibles parmi les patients atteints de la MA avec MCEREV que parmi ceux atteints seulement de la MA (p<0.01), mais les différences absolues étaient très faibles (84% vs. 86% pour l’utilisation continue de ChEIs ; 77% vs. 78% pour la thérapie avec ChEIs interrompue). Pour les critères d’évaluation secondaires, la période jusqu’au décès était plus courte chez les patients avec la MCEREV que sans la MCEREV, mais la période jusqu’au vi placement en hebergement n’était pas différente entre les deux groupes. Dans l'analyse primaire (non-biaisée), aucune association a été trouvée entre le type de ChEI et la mort ou le placement en maison d'hébergement. Cependant, après l'introduction du biais de temps-immortel, on a observé un fort effet différentiel. Limitations: Les résultats peuvent avoir été affectés par le biais de sélection (classification impropre), par les différences entre les groupes en termes de consommation de tabac et d’indice de masse corporelle (ces informations n’étaient pas disponibles dans les bases de données de la RAMQ) et de durée de la thérapie avec les ChEIs. Conclusions: Les associations entre la coexistence d’une MCEREV et la durée de la période jusqu’au placement en hebergement ou au décès apparaissent peu pertinentes cliniquement parmi les patients atteints de la MA traités avec des ChEIs. L’absence de différence entre les patients atteints de la MA souffrant ou non de la MCEREV suggère que la coexistence d’une MCEREV ne devrait pas être une raison de refuser aux patients atteints de la MA l’accès au traitement avec des ChEIs. Le calcul des « personne-temps » non exposés dans l'analyse élimine les estimations biaisées de l'efficacité des médicaments. / Introduction: Dementia may be caused by Alzheimer’s disease (AD), cerebrovascular disease (CVD), or a combination of both. When CVD is associated with dementia, survival is thought to be reduced. It is unclear whether treatment with cholinesterase inhibitors (ChEIs), which has been found to improve cognitive symptoms and global function in AD patients, has similar benefits in vascular forms of dementia. Objectives: The present study was designed to determine whether co-existing CVD is associated with survival or time to nursing home placement (NHP) among AD patients treated with ChEIs. Findings of poorer outcomes in patients with versus without CVD might argue against the use of ChEIs for AD patients in whom CVD co-exists. The objective of a second analysis was to assess for the first time in patients with AD the potential impact of immortal time (and followup) bias on risk for these outcomes. Methods: A retrospective cohort study was undertaken using the Régie de l’Assurance Maladie du Québec (RAMQ) databases to examine the time to NHP or death for AD patients aged 66+, with or without CVD, treated with ChEIs between July 1, 2000, and June 30, 2003. Because ChEIs are approved only for AD in Canada, a ChEI prescription was used as a surrogate for an AD diagnosis. Concomitant CVD was identified on the basis of a lifetime diagnosis of stroke or ii endarterectomy, or a diagnosis of transient ischemic attack within the six months prior to the index date. Separate analyses were performed for patients with persistent ChEI use and those who discontinued ChEI therapy. Seven Cox proportional hazard regression models which varied in the definition of the index date (start of follow-up) and the duration of follow-up were used to evaluate the impact of immortal time bias. Results: 4,428 patients met inclusion criteria for AD with CVD; 13,512 were classified as having AD alone. For the composite endpoint of NHP or death, 1,000-day survival rates were lower among AD patients with versus without CVD (p<0.01), but absolute differences were very small (84% vs. 86% with continuous ChEI use; 77% vs. 78% with discontinuous ChEI therapy). Of the secondary endpoints, time to death was shorter for patients with versus without CVD, but time to NHP did not differ between groups. In the primary, unbiased analysis, no association was found between ChEI treatment type and death or NHP. However, after introduction of immortal time bias, a strong differential effect was observed. Limitations: Results may have been affected by selection (misclassification) bias, between-group differences in smoking and body mass index (information on which was not available in the RAMQ databases), and duration of ChEI therapy. Conclusions: Associations between co-existing CVD and time to NHP or death appeared to be of little clinical relevance among AD patients treated with ChEIs. iii The lack of difference between AD patients with and without CVD suggests that CVD should not be used as a reason to deny AD patients access to ChEI treatment. Properly accounting for unexposed person-time in the analysis eliminates biased estimates of drug efficacy.

In this thesis study we examined glycogen synthase kinase-3β (GSK-3β) and its effects over Nrf2 and Pin 1 as it relates to Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized by a prolonged high oxidative environment. Transcription factor Nrf2 is vital in the brain’s defense against oxidative insults through its up-regulation of over 100 antioxidants. Depletion of the brain’s antioxidant defense system results in intolerance to an oxidative environment, contributing to the progression of AD. The regulatory Pin 1 protein promotes cellular homeostasis, and when down-regulated results in increased deposits of neurofibrillary tangles (NFTs) and amyloid-β (Aβ) plaques, the two pathological hallmarks of AD. Using aged SAMP8 mice treated with antisense oligonucleotide (AO) directed at GSK-3β and random AO, the data presented here demonstrate decreased oxidative stress and increased Nrf2 transcriptional activity and Pin 1 levels as a result of the down-regulation of GSK-3β. Collectively, these results implicate GSK-3β activity in the increased oxidative stress of AD and support its inhibition as a possible therapeutic treatment for the disease. Further, we elucidate a possible mechanism connecting GSK-3β to the loss of tolerance to an oxidative environment and increased deposits of NFTs and Aβ plaques observed in AD.

Today, as we live longer, dementia diseases are becoming more prevalent around the world. Thus, further knowledge of how to maintain levels of cognitive functioning in old age and how to identify factors that postpone the onset of dementia are of acute interest. Lifestyle patterns and social life are important aspects to consider in this regard. This thesis includes three studies. Study I investigated the association between participation in various leisure activities in old age (≥65 years) and risk of incident all-cause dementia. Analyses of the total follow-up time period (15 years) showed that higher levels of “Social” and “Total” leisure activity were associated with decreased risk of dementia. In Study II, the aim was to investigate the association between various aspects of social relationships in old age (≥65 years) and risk of incidents of all-cause dementia and Alzheimer's disease. Results showed that over the total follow-up period (16 years) higher values on the relationship index were associated with reduced risk of both dementia and Alzheimer's disease. Visiting/visits of friends and acquaintances more than once a week was related to decreased risk for all-cause dementia, but not for Alzheimer's disease. However, in neither Study I nor II did any of these factors alter the risk of all-cause dementia or Alzheimer's disease when near-onset dementias were removed from the analyses (Study I, up to five years; Study II, up to three years). In Study III the aim was to investigate the association between social network size and cognitive ability in a middle-aged (40–60 years) sample. The idea was that if social network size can moderate negative age-related influence on memory functions, it might also put an individual on a cognitive trajectory that is beneficial in old age. Results from longitudinal analyses showed that baseline network size was positively related to five-year changes in semantic memory and with changes in both semantic and episodic memory at the ten-year follow-up. Social network size was unrelated to changes in visuospatial performance. Taken together, enrichment factors measured in old age (≥ 65 years) did not alter the risk of all-cause dementia or Alzheimer's disease when near-onset dementias were removed from the analyses. These results might reflect protective short-term effects or reverse causality, meaning that in the prodromal phase of dementia individuals tend to withdraw from activity. Social network size in middle age (40-60 years), however, appears to have beneficial long-term effects on cognitive functioning. The results highlight the importance of long follow-up periods and the need to adjust for the influences of reverse causality when investigating the impact of a socially and mentally active life on cognitive functioning.

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values &lt; 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Objective: To study awareness of cognitive dysfunction in patients with very mild Alzheimer’s disease (AD) and subjects with mild cognitive impairment (MCI). Methods: A complaint interview covering 13 cognitive domains was administered to 82 AD and 79 MCI patients and their caregivers. The patient groups were comparable according to age and education, and Mini Mental State Examination (MMSE) scores were ≥24 in all cases. The discrepancy between the patients’ and caregivers’ estimations of impairments was taken as a measure of anosognosia. Results: Self-reports of cognitive difficulties were comparable for AD and MCI patients. However, while in comparison to caregivers MCI patients reported significantly more cognitive impairment (p < 0.05), AD patients complained significantly less cognitive dysfunctions (p < 0.001). Conclusions: While most MCI patients tend to overestimate cognitive deficits when compared to their caregiver’s assessment, AD patients in early stages of disease underestimate cognitive dysfunctions. Anosognosia can thus be regarded as a characteristic symptom at a stage of very mild AD (MMSE ≥24) but not MCI. Accordingly, medical history even in mildly affected patients should always include information from both patient and caregiver. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.