Discovery of Novel Serum Biomarkers for Diagnosing and Staging Alzheimer’s DiseaseDipti Jigar ShahDepartment of Chemistry and Biochemistry, BYUDoctor of PhilosophyAlzheimer’s disease (AD) is an untreatable neurologic disease affecting more than 5 million Americans, most over 60 years of age. Protein plaques and neurofibrillary tangles typify AD brain pathology and are thought to cause the progressive dementia and brain shrinkage observed in AD. Currently there are no methods to diagnose the disease at a time before damage becomes irreversible.Biochemical tests for AD using cerebrospinal fluid analysis or neuroimaging are not yet sufficiently sensitive and specific, and they are invasive. This points to a need for a more easily applied and more sensitive diagnostic test. Although the gross anatomical changes are localized to the brain, AD is likely to involve changes throughout the body. As a result of this, changes in the abundance of certain biomolecules present in the circulation system are likely to occur. Consequently, a serum proteomics approach able to measure such changes, when applied to AD, would likely find quantitative changes in relevant molecules that can help diagnose the disease correctly, ideally early in the disease process. The goal of this work was to discover and validate novel diagnostic serum biomarkers for AD. For biomarker discovery and validation, we used a novel serum proteomics approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry. Our samples were protein depleted, which helped us survey low molecular weight species in the serum without ion suppression from larger proteins like albumin. We were able to observe more than 8000 molecular species in a single run. The overall project was comprised of four studies: (i) discovery of novel potential serum AD markers, (ii) blinded validation of diagnostically promising biomarkers found in the initial study, with their further chemical identification, (iii) exploring gender-based serum AD biomarkers, and (v) discovery of biomarkers that distinguish early versus moderate stage AD. In the first study, the approach found 38 significant (p < 0.05) biomarkers and 21 near significant (p = 0.05 to 0.099) biomarkers. On using the forward selection approach, we built multi-marker panels with specificities and sensitivities higher than 80%.The second study reports on a blinded validation study that was performed on a new set of serum samples. We focused on the 13 most promising AD biomarkers found as part of the initial study. We successfully validated 4 of these biomarkers that showed highly significant statistical p-values. As part of this study, research was conducted to identify these 4 biomarkers, which was accomplished using tandem mass spectrometry with fragmentation experiments. The third study used data from the initial study but looked at gender specific biomarkers. We found 31 significant and near significant serum AD biomarkers for women, 16 for men, and 25 that were gender independent. Multi-marker panels of AD biomarkers for women or men had sensitivities of >60% and specificities >85%.In the fourth study, cases with moderate AD were compared to cases with very mild or mild AD to find novel biomarkers that could be used for staging. We found 44 significant and near significant biomarkers that were quantitatively different between mild and severe AD. In conclusion, we were successful in accomplishing the goal of this work of finding, validating and identifying novel serum biomarkers that diagnose AD.

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This critical synopsis of prior work by Clive Beggs is submitted in support of a PhD by published work. The work focuses on venous and cerebrospinal fluid (CSF) anomalies associated with multiple sclerosis (MS) and other neurological diseases. MS is characterized by focal inflammatory lesions, which are often venocentric. Recently a vascular syndrome, chronic cerebrospinal venous insufficiency (CCSVI) has been linked with MS. This syndrome, which is characterized by constricted cerebral venous outflow, has become mired in controversy, with various studies producing conflicting findings, with the result that the science associated with CCSVI has become obscured. Clive Beggs work seeks to bring clarity to the debate surrounding CCSVI by characterizing physiological changes associated with constricted cerebral venous outflow. The work submitted here involves collaborative studies with Robert Zivadinov (University of Buffalo), Paolo Zamboni (University of Ferrara), and Chih- Ping Chung (National Yang Ming University of Medicine). The key findings of these studies are: (i) MS patients, diagnosed with CCSVI, exhibit greatly increased hydraulic resistance of the cerebral venous drainage system; (ii) MS patients experience loss of the small cerebral veins; (iii) MS patients exhibit reduced CSF bulk flow, consistent with mild venous hypertension; (iv) MS patients exhibit increased CSF pulsatility in the Aqueduct of Sylvius, which appears to be linked with mild venous hypertension associated with CCSVI; and (v) jugular venous reflux is associated with white matter and parenchymal volumetric changes in Alzheimer’s patients. Collectively, these findings suggest that extracranial venous anomalies are associated with changes in the intracranial physiology.

Alzheimer’s disease (AD) drugs in therapy are limited to acetylcholine esterase inhibitors and memantine. Newly developed drugs against a single target structure have an insufficient effect on symptomatic AD patients. Results: Novel aromatically anellated pyridofuranes have been evaluated for inhibition of AD-relevant protein kinases cdk1, cdk2, gsk-3b and Fyn. Best activities have been found for naphthopyridofuranes with a hydroxyl function as part of the 5-substituent and a hydrogen or halogen substituent in the 8-position. Best results in nanomolar ranges were found for benzopyridofuranes with a 6-hydroxy and a 3-alkoxy substitution or an exclusive 6-alkoxy substituent. Conclusion: First lead compounds were identified inhibiting two to three kinases in nanomolar ranges to be qualified as an innovative approach for AD multitargeting.

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Extracellular accumulation of Aβ in β-amyloid plaques is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients, although a lack of correlation with cognitive decline raised doubts on this hypothesis. In different transgenic mouse models Aβ accumulates inside the cells and mice develop behavioral deficits well before visible extracellular β-amyloid accumulation. Here we show that intracellular Aβ accumulates in flotillin-1 positive endocytic vesicles. We also demonstrate that flotillin-1 is not only associated with intracellular Aβ in transgenic mice but also with extracellular β-amyloid plaques in AD patient brain sections. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La maladie d’Alzheimer est majoritairement sporadique et peu est connu sur les mécanismes déclenchant le développement de cette forme de la maladie. Les études sur la forme familiale ont attribué une importance particulière à la bêta-amyloïde (Aβ), un produit de clivage du précurseur de la protéine amyloïde (APP). Plusieurs facteurs de risques ont été identifiés comme déclencheurs potentiels du développement de la maladie d’Alzheimer dont le diabète de type II (T2D). En effet, la déficience de la signalisation de l’insuline par la désensibilisation des récepteurs de l’insuline (IR) dans le cerveau semble être une caractéristique commune aux deux maladies. Les effets à long terme de la résistance à l’insuline sur l’accumulation d’Aβ et sur la phosphorylation de Tau ont été étudiés, mais les effets de la perturbation aiguë des IR sont moins bien caractérisés. Aussi, les désordres métaboliques sont également des caractéristiques communes aux deux maladies. Le but de notre étude est de déterminer si la perturbation aiguë des IR peut affecter le clivage de l’APP et si la privation énergétique par le jeûne peut sensibiliser ce clivage à la perturbation aiguë des IR. Pour évaluer ceci, nous avons utilisé la Tyrphostin AG1024 à faible dose pour simuler une perturbation des IR plutôt qu’un blocage complet des récepteurs. Pour quantifié le clivage de l’APP, nous avons mesuré les changements de la quantité d’APP taille pleine totale par immunobuvardage de type Western. Pour s’assurer que les changements de la quantité d’APP taille pleine traduisait bien un clivage, nous avons développé un système de lecture par luciférase. Ce système nous permet de suivre le clivage de l’APP via l’expression de la luciférase Firefly puisque le facteur de transcription GAL4 est lié à la portion C-terminale de l’APP . Tout d’abord, nous avons observé que la perturbation aiguë des IR par la Tyrphostin mène au clivage de l’APP et que le jeûne augmente la vulnérabilité au clivage de l’APP suite à une plus petite dose de Tyrphostin. Ce clivage serait imputable à la voie amyloïdogénique puisque l’inhibition de la β-secrétase et de la γ-secrétase empêche le clivage de l’APP. Nous avons aussi montré que la perturbation des IR est nécessaire alors que la perturbation spécifique des IGF-1R n’est pas suffisante. De plus, ni l’autophagie, ni les caspases et ni le protéasomes ne semblent impliqués dans le clivage de l’APP suivant la combinaison du jeûne et de la petite perturbation des IR. L’activité de mTOR n’est également pas requise. Cependant, l’activité de la GSK3 est nécessaire au clivage et semble affecter le clivage par la γ-secrétase. Nous avons ensuite confirmé dans des cultures primaires neuronales que la combinaison du jeûne et de la perturbation aiguë des IR cause le clivage de l’APP et que la GSK3 est encore une fois fortement active. Ainsi, nos résultats suggèrent que la perturbation de la signalisation de l’insuline tel qu’observé dans le T2D augmente le clivage de l’APP via la voie amyloïdogénique et, donc, contribue à la pathologie de la maladie d’Alzheimer. / Little is known about the mechanisms that trigger the onset of Alzheimer’s disease (AD), a primarily sporadic disease. Studies on the familial form of AD attributed a particular importance to Amyloid beta (Aβ), a cleavage product of the Amyloid precursor protein (APP). Many risk factors have been identified as potential triggers of the development of AD including Type 2 diabetes (T2D). Indeed, the impairment of insulin signaling by the desensitization of insulin receptors (IR) in the brain seems to be a common hallmark of both diseases. The long term effects of IR resistance on the accumulation of Aβ and Tau hyperphosphorylation have been studied, but the acute effects of IR perturbation is less characterized. Also, both diseases show metabolic defects. Our research aimed to determine whether acute perturbation of IR signaling affects APP processing and if starving (energy deprivation) could sensitize this processing to acute perturbation of IR. To assess this, we used small doses of Tyrphostin AG1024 to simulate IR perturbations rather than a complete blocakade of the receptors. To quantify APP processing, we measured the change of total full- lenght APP by Western blot. To ensure that this change reflected APP processing we developed a luciferase based readout system. This system allowed us to monitor the occurrence of APP cleavage via GAL4-UAS Firefly luciferase driven expression because we linked GAL4 transcription factor to the C-terminal region of APP. First, we showed that IR perturbation with Tyrphostin leads to APP processing and that starving increased IR susceptibility to a smaller doses of Tyrphostin. This APP processing occurs via the amyloidogenic pathway because inhibition of β- and γ-secretase inhibited APP processing. We showed that this processing absolutely requires IR perturbation, while IGF-1R perturbation alone is insufficient. Furthermore, neither autophagy, caspases nor proteasome seemed to be implicated in APP processing following starvation and small IR perturbation. The activity of mTOR is also not required. On the contrary, GSK3 activation is necessary for the processing and seems to affect γ-secretase cleavage. We then confirmed in primary cultured neurons that the combination of acute starvation and small IR perturbation causes APP cleavage and GSK3 is again strongly activated. Taken together, our results suggest that the impairment of IR signalling seen in T2D increases the processing of APP via the amyloidogenic pathway and thereby contributes to the pathology of AD.

Background: Subjective cognitive decline (SCD), i.e., the self-perceived feeling of worsening cognitive function, may be the first notable syndrome of preclinical Alzheimer’s disease and other dementias. However, not all individuals with SCD progress. Stability of SCD, i.e., repeated reports of SCD, could contribute to identify individuals at risk, as stable SCD may more likely reflect the continuous neurodegenerative process of Alzheimer’s and other dementias. Methods: Cox regression analyses were used to assess the association between stability of SCD and progression to MCI and dementia in data derived from the population-based Leipzig Longitudinal Study of the Aged (LEILA75+). Results: Of 453 cognitively unimpaired individuals with a mean age of 80.5 years (SD = 4.2), 139 (30.7 %) reported SCD at baseline. Over the study period (M = 4.8 years, SD = 2.2), 84 (18.5 %) individuals had stable SCD, 195 (43.1 %) unstable SCD and 174 (38.4 %) never reported SCD. Stable SCD was associated with increased risk of progression to MCI and dementia (unadjusted HR = 1.8, 95 % CI = 1.2–2.6; p < .01), whereas unstable SCD yielded a decreased progression risk (unadjusted HR = 0.5, 95 % CI = 0.4–0.7; p < .001) compared to no SCD. When adjusted for baseline cognitive functioning, progression risk in individuals with stable SCD was significantly increased in comparison to individuals with unstable SCD, but not compared to individuals without SCD. Conclusions: Our results, though preliminary, suggest that stable SCD, i.e., repeated reports of SCD, may yield an increased risk of progression to MCI and dementia compared to unstable SCD. Baseline cognitive scores, though within a normal range, seem to be a driver of progression in stable SCD. Future research is warranted to investigate whether stability could hold as a SCD research feature.

Alzheimer’s disease (AD) is the leading cause of dementia and is alone responsible for 60-70% of all cases of dementia. Though sharing clinical symptoms with other types of dementia, the hallmarks of AD are the abundance of extracellular depositions of β-amyloid (Aβ) plaques, intracellular neurofibrillary tangles of hyper phosphorylated tau proteins and synaptic depletion. The onset of the physiological hallmarks may precede clinical symptoms with a decade or more, and once clinical symptoms occur it may be difficult to separate AD from other types of dementia based on clinical symptoms alone. Since the introduction of radiolabeled Aβ tracer substances for positron emission tomography (PET) imaging it is possible to image the Aβ depositions in-vivo, strengthening the confidence in the diagnosis. Because the accumulation of Aβ may occur years before the first clinical symptoms are shown and even reach a plateau, Aβ PET imaging may not be feasible for disease progress monitoring. However, a negative scan may be used to rule out AD as the underlying cause to the clinical symptoms. It may also be used as a predictor to evaluate the risk of developing AD in patients with mild cognitive impairment (MCI) as well as monitoring potential effects of anti-amyloid drugs.Though currently validated for dichotomous visual assessment only, there is evidence to suggest that quantification of Aβ PET images may reduce inter-reader variability and aid in the monitoring of treatment effects from anti-amyloid drugs.The aim of this thesis was to refine existing methods and develop new ones for processing, quantification and visualization of Aβ PET images to aid in the diagnosis and monitoring of potential treatment of AD in clinical routine. Specifically, the focus for this thesis has been to find a way to fully automatically quantify and visualize a patient’s Aβ PET image in such way that it is presented in a uniform way and show how it relates to what is considered normal. To achieve the aim of the thesis registration algorithms, providing the means to register a patient’s Aβ PET image to a common stereotactic space avoiding the bias of different uptake patterns for Aβ- and Aβ+ images, a suitable region atlas and a 3-dimensional stereotactic surface projections (3D SSP) method, capable of projecting cortical activity onto the surface of a 3D model of the brain without sampling white matter, were developed and evaluated.The material for development and testing comprised 724 individual amyloid PET brain images from six distinct cohorts, ranging from healthy volunteers to definite AD. The new methods could be implemented in a fully automated workflow and were found to be highly accurate, when tested by comparisons to Standards of Truth, such as defining regional uptake from PET images co-registered to magnetic resonance images, post-mortem histopathology and the visual consensus diagnosis of imaging experts.

Orientador: Andriana Susana Lopes de Oliveira Campanharo / Resumo: O Alzheimer é uma doença degenerativa do cérebro, incurável, que se agrava ao longo do tempo e atinge sobretudo pessoas entre 65 e 90 anos. A doença de Alzheimer (DA) é a principal demência entre os idosos e caracteriza-se por perda de funções cognitivas (memória, orientação, comportamento e linguagem), causada pela morte de células cerebrais. Atualmente, o diagnóstico definitivo da DA é feito por meio de um exame do tecido cerebral obtido por biopsia ou necropsia. Como somente após a morte do paciente pode-se ter a certeza que o mesmo tinha a DA, seu diagnóstico é feito utilizando exames, avaliações e excluindo-se outras causas de demência do seu histórico clínico. Em paralelo, estudos têm sido desenvolvidos para o estudo da DA com base de dados de EletroEncefaloGrama (EEG), e nesse sentido, diversos métodos de análise de dados de EEG têm sido propostos. Contudo, o estudo da DA por meio de dados de EEG é ainda um desafio, e consequentemente, faz-se necessária a preposição de novos métodos com o intuito de capturar informações adicionais da doença. Nesse sentido, utilizamos o mapeamento de uma série temporal em uma rede complexa no estudo da dinâmica de séries temporais de EEG de pacientes com a DA. Mais especificamente, na distinção entre envelhecimento e a DA e na identificação, dos estágios da DA em pacientes doentes a partir, das ondas cerebrais, alfa, beta, teta e delta. / Abstract: Alzheimer’s is a degerative brain disease, incurable, which aggravates over time and mainly affects people between 65 and 90 years. Alzheimer’s disease (AD) is the leading dementia among the elderly and is characterized by cognitive functions loss (memory, orientation, behavior and language) caused by the death of brain cells. Currently, confirmatory diagnosis of AD can only be made through the examination the brain tissue obtained by biopsy or necropsy. Considering that only after the patient’s death it is possible to be sure that he or she had AD, the approximate diagnosis is made excluding other causes of dementia. In parallel, studies have been developed for the study of AD with the use of Electroencephalography (EEG), and in this sense, several methods of EEG data analysis have been proposed. However, the study of AD with the use of EEG data is still a challenge, and consequently, it is necessary the proposal of new methods to capture additional information about the disease. In this sense, we used the map from a time series to a network, recently proposed by Campanharo, in a novel application, that is, in the study of EEG time series of patients with AD. More specifically, to distinguish aging and AD and to identify the stages of AD in unhealthy patients based on alpha, beta, theta and delta brain waves information. / Mestre